CN104840944A - Methods for increasing thermogenic adipocytes - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for increasing thermogenic adipocytes (e.g., brown adipocytes or other UCP-1 expressing adipocytes) by administering an antagonist of an ActRIIB signaling pathway. Examples of such antagonists include ActRIIB polypeptides, anti-ActRIIB antibodies, anti-myostatin antibodies, anti-GDF3 antibodies, anti-Nodal, anti-activin, and anti-GDF11 antibodies. A variety of metabolic and other disorders may be treated by causing an increase in thermogenic adipocytes.

Description

For increasing the method for heat production adipose cell

The application is divisional application, and the applying date of original application is on June 8th, 2010, and application number is 201080036063.2 (PCT/US2010/037779), and denomination of invention is " method for increasing heat production adipose cell ".

The cross reference of related application

This application claims the rights and interests of following U.S.Provisional Serial: 61/280,545 of the application in 3,61/276,422 and 2009 on November that in the JIUYUE, 61/268,128,2009 of applying on June 8th, 2009 are applied on the 10th.The instruction of all above-mentioned applications of quoting is incorporated herein by reference.

Background of invention

Mammalian adipocyte is categorized as the white adipocyte of storage power or catabiotic brown fat cell traditionally.Brown fat cellular expression Uncoupling pro-tein-2 (UCP1), it produces and mitochondrion proton gradient uncoupling and be heat (Cannon etc., 2004, Physiol Rev 84:277-359) by biochemistry power conversion by making ATP.Such heat production is used for maintaining body temperature under cold environmental condition, or promotes the energy balance when in the face of excessive energy intake.In order to emphasize the metabolic importance of brown fat, in mice, make the severe obesity that its genetic defect causes insulin resistant, hyperglycemia, hyperlipemia and hypercholesterolemia adjoint.(Lowell etc., 1993, Nature 366:740-742; Hamann etc., 1995, Diabetes44:1266-1273; Hamann etc., 1996, Endocrinology 137:21-29).In view of UCP-1 is as the effect of important uncoupling protein, the adipose cell of expressing UCP-1 will have Thermogenic Activity.

In the mankind, brown adipose tissue plays important calorigenic action in baby, but shrinks between the period of development after birth, and thinks rare and inessential clinically in adult in history.But this idea has been overturned in nearest discovery, and sizable interest is created to the effect of brown adipose tissue between the manhood.Particularly, positron emission tomography and computed tomography (PET-CT) are combined and causes brown fat storehouse (Nedergaard etc. that are active at adult's camber of remarkable percentage ratio, presumption accidentally being detected for monitoring neoplasm metastasis, 2007, Am J Physiol Endocrinol Metab 293:E444-E452).Research subsequently confirms in health adult, these storehouses are really for expressing the function brown fat (Virtanen etc. of UCP1,2009, N Engl J Med 360:1518-1525), in the young men of the research more than 90%, during cold expoure, observe brown adipose tissue activity, but during neutral condition, then do not observe (van Marken Lichtenbelt etc., 2009, N Engl J Med 360:1500-1508).In addition, the amount that the retrospective analysis of the nearly 2,000 PET-CT scannings thus carried out due to different Diagnosis of Primary shows active brown fat and Body Mass Index (a kind of tolerance of widely used overall obesity) inversely related, cause the probability (Cypess etc. of brown fat important beneficial effect in adult metabolism, 2009, N Engl J Med 360:1509-1517).Not clear is is dispersed in the effect of heat production adipose cell in white adipose tissue (such as, brown fat cell or other express the adipose cell of UCP-1).

In view of the important metabolic activity of heat production adipose cell, in body, increase the needs of the material of (activity such as by being formed and/or increasing) heat production adipose cell.

Summary of the invention

In some respects, this description is provided for the method by using the antagonist of ActRIIB signal transduction pathway to increase heat production adipose cell in patient.Such antagonist can be, such as solubility ActRIIB albumen (such as ActRIIB-Fc fusion rotein), be combined or suppress the antagonist that ActRIIB expresses with ActRIIB, and with carried out the ligand binding of signal transduction by ActRIIB or suppress it to express and participate in the antagonist of the adjustment of heat production adipose cell.Such part comprises muscle mass (myostatin, i.e. GDF8), GDF3, activator protein (such as activator protein A, B, C or E), GDF11 and Nodal.

In some respects, this description ActRIIB related polypeptide be provided for by giving patient effective amounts in need increases the method for heat production adipose cell.ActRIIB related polypeptide can be the ActRIIB polypeptide (such as ActRIIB ectodomain or its part) be combined with ActRIIB part such as GDF3, GDF8, GDF11, activator protein or Nodal.Optionally, the Kd of ActRIIB polypeptide and ActRIIB ligand binding is less than 10 micro-molar concentrations, or is less than 1 micro-molar concentration, 100,10 or 1 nanomolar concentration.Multiple suitable ActRIIB polypeptide describes in following open PCT patent application, and all open PCT patent applications are incorporated herein by reference: WO 00/43781, WO 04/039948, WO 06/012627, WO 07/053775, WO 08/097541 and WO 08/109167.Optionally, ActRIIB polypeptide suppresses ActRIIB signal transduction, the intracellular signal transduction event such as caused by ActRIIB part.The solubility ActRIIB polypeptide used in this preparation can be any polypeptide disclosed herein, such as have be selected from SEQ ID NO:1,2,5,6,12, the polypeptide of the aminoacid sequence of 14 and 17, or have be selected from SEQ ID NO:1,2,5,6,12, the aminoacid sequence of 14 and 17 has the polypeptide of the aminoacid sequence of at least 80%, 85%, 90%, 95%, 97% or 99% homogeneity.Solubility ActRIIB polypeptide can comprise the function fragment of natural A ctRIIB polypeptide, such as comprise be selected from SEQ ID NO:1,2,5,6,12, at least 10,20 or 30 amino acid whose polypeptide of the sequence of 14 and 17, or comprise the polypeptide of sequence (disappearance C-terminal 1,2,3,4,5 or 10-15 aminoacid and lack 1,2,3,4 or 5 aminoacid at N-terminal) of SEQ ID NO:1.Optionally, polypeptide will comprise and will be no more than 3 amino acid whose truncates relative to SEQ ID NO:1 at a N-terminal 2-5 aminoacid with at C-terminal.Another polypeptide shows for SEQ ID NO:12.Solubility ActRIIB polypeptide can (such as in ligand binding domains) comprise and changes relative to one, two, three, four, five or more of naturally occurring ActRIIB polypeptide in aminoacid sequence.Relative to naturally occurring ActRIIB polypeptide, when the change in aminoacid sequence can such as produce in mammal, insecticide or other eukaryotic cells, change the glycosylation of polypeptide, or change the proteolytic cleavage of polypeptide.Solubility ActRIIB polypeptide can be fusion rotein, its have as a domain ActRIIB polypeptide (ligand binding domains of such as ActRIIB or its variant) and the one or more extra domain of required character (pharmacokinetics such as improved, easier purification, targeting particular organization etc.) is provided.Such as, the domain of fusion rotein can strengthen one or more following character: half life, absorption/administration, tissue positioned or distribution, the formation of protein complexes, the multimerization of fusion rotein and/or purification in body internal stability, body.Solubility ActRIIB fusion rotein can comprise immunoglobulin constant domains, such as Fc domain (wild type or mutant) or serum albumin.In specific embodiments, ActRIIB-Fc fusion rotein comprises the relatively non-structured joint between Fc domain and the outer ActRIIB domain of born of the same parents.This destructuring joint may correspond to about 15 amino acid whose destructuring regions (" tail ") of the ectodomain C-terminal in ActRIIB, or it can be relatively containing secondary structure have 5-15,20,30,50 or more an amino acid whose artificial sequence.Joint can be rich in glycine and proline residue, and such as can comprise the repetitive sequence of threonine/serine and glycine, such as TG ₄repeat (SEQ ID NO:18) or SG ₄repeat (SEQ ID NO:19).Fusion rotein can comprise purification subsequence (subsequence), such as epitope tag, FLAG label, polyhistidine sequence and GST fusant.Optionally, solubility ActRIIB polypeptide comprises and is one or morely selected from following modified amino acid residue: glycosylated amino acid, PEGization aminoacid, farnesylated amino acid, acetylated amino acids, biotinylation aminoacid, the aminoacid puted together with lipid part and the aminoacid puted together with organic derivatizing agents (derivatizing agent).Usually preferably in following mammal cell line, ActRIIB albumen is expressed: it suitably mediates the Natively glycosylated of ActRIIB albumen to reduce unfavorable immunoreactive probability in patient.Successful Application people and Chinese hamster ovary celI system, and expect that other common mammalian expression vectors are useful.

In some respects, compound disclosed herein can be mixed with pharmaceutical preparation.Pharmaceutical preparation also can comprise one or more other compounds, such as, be used for the treatment of the compound of ActRIIB relevant disease.Preferably, pharmaceutical preparation is substantially free of pyrogen.

In some respects, this description provides the encoding soluble ActRIIB nucleic acid of polypeptide, its ActRIIB polypeptide of not encoding complete.The polynucleotide be separated can comprise the coded sequence of solubility ActRIIB polypeptide such as mentioned above.Such as, the nucleic acid be separated can comprise the sequence of the ectodomain (such as ligand binding domains) of coding ActRIIB, with part or all of membrane spaning domain and/or the cytoplasmic domains of coding ActRIIB, but the sequence of the termination codon in membrane spaning domain or cytoplasmic domains or between ectodomain and membrane spaning domain or cytoplasmic domains of not encoding.Such as, the polynucleotide be separated can comprise total length ActRIIB polynucleotide sequence such as SEQ ID NO:4, or part clipped form, the polynucleotide of described separation also comprise the translation stop codon before 3 ' end at least 600 nucleotide, or are positioned at other position and make the translation of polynucleotide produce the translation stop codon of the ectodomain of the truncate partial fusion of optional and total length ActRIIB.Nucleic acid disclosed herein effectively can be connected with the promoter for expressing, and this description provides the cell transformed with such recombination of polynucleotide.Preferred cell is mammalian cell, such as Chinese hamster ovary celI.

In some respects, this description is provided for the method preparing solubility ActRIIB polypeptide.Such method can be included in suitable cell such as Chinese hamster ovary (CHO) cells any nucleic acid disclosed herein (such as SEQ ID NO:3).Such method can comprise: a) cultured cell under the condition of applicable solubility ActRIIB expression of polypeptides, and wherein said cell solubility ActRIIB expression construct transforms; And b) reclaim the solubility ActRIIB polypeptide of so expressing.Any known technology obtaining albumen from cell culture can be used, solubility ActRIIB polypeptide is recovered as thick, partially purified or highly purified part.

In some respects, use compound as herein described to increase heat production adipose cell to can be used for controlling wherein to control metabolic activity being useful various diseases.Example comprises obesity controlling, reduces body fat content or reduces body fat content advances the speed, and disease therapy such as obesity, noninsulindependent diabetes (NIDDM), type 2 diabetes mellitus, cardiovascular disease, cancer, hypertension, apoplexy, respiratory disorder (respiratory problem), dyslipidemia, lipodystrophy, glucocorticoid administration sequela and gallbladder disease.

In some respects, solubility ActRIIB polypeptide disclosed herein can use in the method being used for the treatment of the experimenter suffering from muscle loss or the not enough relevant disease of muscle growth, wherein such disease is also relevant to metabolic disease, such as obesity, lipodystrophy, diabetes (such as type ii diabetes), cachexia or other above-mentioned diseases.Such disease comprises muscular dystrophy, muscle decay syndrome (sarcopenia) and HIV (it can be relevant with lipodystrophy to amyotrophy (muscle wasting)).

In some respects, this description is provided for the method for the activity of ActRIIB polypeptide or ActRIIB part (such as GDF8, GDF11, activator protein, GDF3 and Nodal) in antagonize cellular.The method comprises and being contacted with solubility ActRIIB polypeptide by cell.Optionally, the signal transduction that the activity of ActRIIB polypeptide or ActRIIB part is mediated by ActRIIB/ActRIIB ligand complexes is monitored, such as, by monitoring cell proliferation or UCP-1 expression.The cell of the method comprises osteoblast, chondrocyte, myogenous cell, adipose cell and myocyte.

In some respects, this description provides the purposes of solubility ActRIIB polypeptide in the medicine for the preparation for the treatment of disease as herein described or disease.

In some respects, this description is provided for the method increasing heat production adipose cell in patient in need, such method can comprise give effective dose be selected from following compound: comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with the sequence of the aminoacid 29-109 of SEQ ID NO:2 and by under stringent hybridization condition with the polypeptide of the nucleic acid coding of the nucleic acid hybridization of SEQ ID NO:3.Polypeptide can be the fusion rotein comprising heterologous moiety.Polypeptide can be dimer.Polypeptide can merge with the constant domain of immunoglobulin.Polypeptide can with the Fc partial fusion of immunoglobulin such as IgG1, IgG2, IgG3 or IgG4.Polypeptide can comprise the aminoacid sequence having at least 80%, 90%, 93%, 95%, 97%, 98%, 99% or 100% homogeneity with the sequence of aminoacid 29-109,29-128,29-131,29-134,25-109,25-128,25-131,25-134 or 20-134 of SEQ ID NO:2.Polypeptide can comprise with SEQ ID NO:5,6,12, the amino acid whose sequence of 14 or 17 has the aminoacid sequence of at least 80%, 90%, 93%, 95%, 97%, 98%, 99% or 100% homogeneity.Can be the patient suffering from disease described herein with the patient of such compounds for treating, described disease comprises such as metabolic disease (such as obesity, diabetes, metabolism syndrome, dyslipidemia or lipodystrophy) or the muscle disease relevant to metabolic disease (such as muscle decay syndromic certain situation).The administration of compound can promote that the UCP-1 of (optionally in white adipose tissue) in the adipose cell for the treatment of patient expresses.

In some respects, this description is provided for the method increasing heat production adipose cell in patient in need, the method comprises the compound giving effective dose, and the part that this compound carries out signal transduction by targeting ActRIIB or targeting by ActRIIB suppresses ActRIIB signal transduction pathway.The example of such compound comprises the antagonist of the antagonist of ActRIIB, the antagonist of muscle mass (i.e. GDF-8), antagonist, the antagonist of GDF-11, the antagonist of Nodal and the GDF3 of activator protein (such as activator protein A, activator protein B, activated protein c or activator protein E).Each above-mentioned antagonist can be and to be combined with such target specificity and to suppress antibody or other albumen (such as antibody (such as monoclonal antibody) or propetide (with regard to muscle mass and GDF3)) of described target.Above-mentioned antagonist also can be the compound of the expression suppressing ActRIIB or part, such as, based on the compound (such as antisensenucleic acids or RNAi nucleic acid) of nucleic acid.Be the patient suffering from disease described herein with the patient of such compounds for treating, described disease comprises such as metabolic disease (such as obesity, diabetes, metabolism syndrome, dyslipidemia or lipodystrophy) or the muscle disease relevant to metabolic disease (such as muscle decay syndromic certain situation).The administration of compound can promote that the UCP-1 of (optionally in white adipose tissue) in the adipose cell for the treatment of patient expresses.

Accompanying drawing is sketched

Fig. 1 shows ActRIIB (20-134)-hFc and treats the impact on Uncoupling pro-tein-2 (UCP-1) mRNA level in-site in the epididymal adipose tissues pad of the male mice that high fat diet is fed in 60 days.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; *, p < 0.05 compared with carrier.ActRIIB (20-134)-hFc causes the increase of nearly 9 times of the mRNA of this selected marker of coding brown fat, therefore shows the rise of the brown fat cell Thermogenesis be extensively distributed in this white adipose storehouse.

The heat production Histological change induced in the epididymis white fat tissue in the mice that high fat diet is fed for 60 days is treated in Fig. 2 display by ActRIIB (25-131)-hFc.All microscope photograph show under identical amplification.H and E (H & E) dyeing shows that ActRIIB (25-131)-hFc reduces multilocular adipose cell (arrow) that the fat of brown fat drips size and induction brown fat and to cluster the ability of characteristic.The immunostaining of non-contiguous slices is disclosed in the induction of kytoplasm widely of UCP1 (green fluorescence) in multilocular adipose cell and unilocular adipose cell.

Fig. 3 shows ActRIIB (25-131)-hFc and treats the impact on UCP1 mRNA level in-site in the epididymis white fat of the mice that high fat diet is fed in 60 days.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; N=6-7/ group; *, p < 0.05.ActRIIB (25-131)-hFc causes the increase of the mRNA 60 times of this selected marker of coding brown fat, therefore shows the rise of Thermogenesis in this white adipose storehouse.

The level of the mRNA of the sirtuin family member SIRT-1 that encodes (silent message regulator 2, homologue 1) is treated in the mouse epididymis white adipose of the function of 60 days in Fig. 4 display as diet and ActRIIB (25-131)-hFc.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; N=7/ group; *, p < 0.05, NS=is not remarkable.In the mice that high fat diet is fed, they, more than 70%, return to and do not have significantly different levels from the mice of fed standard chow by the SIRT-1 mRNA level in-site that ActRIIB (25-131)-hFc increases.

The level of the mRNA of the PGC-1 α that encodes (Peroxisome proliferator activator receptor-γ coactivator-1 α) is treated in the mouse epididymis white adipose of the function of 60 days in Fig. 5 display as diet and ActRIIB (25-131)-hFc.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; N=6-7/ group; * *, p < 0.001.In the mice that high fat diet is fed, they, more than 250%, return to and do not have significantly different levels from the mice of fed standard chow by the PGC-1 α mRNA level in-site that ActRIIB (25-131)-hFc increases.

The level of the mRNA of the Foxo-1 that encodes (comprising the albumen O subfamily-1 of a point jaw (forkhead) frame) is treated in the mouse epididymis white adipose of the function of 60 days in Fig. 6 display as diet and ActRIIB (25-131)-hFc.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; N=7/ group; *, p < 0.01.In the mice that high fat diet is fed, they, more than 90%, return to and do not have significantly different levels from the mice of fed standard chow by the Foxo-1 mRNA level in-site that ActRIIB (25-131)-hFc increases.

The level of adiponectin mRNA in the mouse epididymis white adipose of the function of 60 days is treated in Fig. 7 display as diet and ActRIIB (25-131)-hFc.RT-PCR data (relative unit, RU) are meansigma methods ± SEM; N=7/ group; *, p < 0.05.In the mice that high fat diet is fed, the adiponectin mRNA level in-site that ActRIIB (25-131)-hFc increases is more than 60%, and the concentration of the adiponectin that therefore contributes to circulating in these mices raises.

The serum levels of adiponectin in the mice of the function of 60 days is treated in Fig. 8 display as diet and ActRIIB (25-131)-hFc.ELISA measures and detects all main oligomer isotypes (total adiponectin), and data are meansigma methods ± SEM; N=7-8/ group; *, p < 0.01; * *, p < 0.001.High fat diet feed mice in, ActRIIB (25-131)-hFc increase circulation Adiponectin concentration more than 75%, significantly more than standard diet contrast in concentration.

The serum-concentration of insulin in the mice of the function of 60 days is treated in Fig. 9 display as diet and ActRIIB (25-131)-hFc.Data are meansigma methods ± SEM; N=7-8/ group; *, p < 0.01.In the mice that high fat diet is fed, ActRIIB (25-131)-hFc makes insulin concentration be normalized to the level observed in standard diet contrast.

Figure 10 display treats the photo in brown fat storehouse between the right omoplate in the left and right of the function of 60 days as diet and ActRIIB (25-131)-mFc.High fat diet increases the size in storehouse and makes it bright, and ActRIIB (25-131)-mFc reverses these changes to a great extent.

Figure 11 describes the impact that ActRIIB (25-131)-mFc treats the quality of brown fat between omoplate in the mice fed high fat diet for 60 days.Data are the meansigma methods ± SEM in the storehouse, left and right of combination; * *, p < 0.001.ActRIIB (25-131)-mFc reverses high fat diet to the impact of this brown fat storehouse quality.

Figure 12 describes the impact that ActRIIB (25-131)-mFc treats the density of brown fat between omoplate in the mice fed high fat diet for 60 days, as measured by micro-computed tomography (microCT).Data (meansigma methods ± SEM) are to represent based on the normalizated unit on the occasion of the null value with water of bone mineral hydroxyapatite (HA); Therefore fat value is negative, and the value of white adipose is usually close to-120.**，p＜0.01。ActRIIB (25-131)-mFc thoroughly reverses the impact of high fat diet on the density in this brown fat storehouse.

Figure 13 shows whole aminoacid sequences of ActRIIB (25-131)-hFc (SEQ ID NO:14).The ActRIIB ectodomain (natural residue 25-131) of TPA targeting sequencing (residue 1-22) and truncate respectively has underscore.What highlight is the amino acid whose glutamic acid of N-terminal being disclosed as ripe fusion rotein by order-checking.

Figure 14 code displaying ActRIIB (25-131)-hFc (coding strand SEQ ID NO:15 is shown in top, and complementary strand SEQ ID NO:16 with 3 '-5 ' be shown in bottom) nucleotide sequence.The sequence of coding TPA targeting sequencing (nucleotide 1-66) and ActRIIB ectodomain (nucleotide 73-396) has underscore.The aminoacid sequence that ActRIIB (25-131) is corresponding is also shown.

Describe in detail

1. summarize

Mammalian adipocyte can be categorized as the white adipocyte of storage power or catabiotic brown fat cell.Uncoupling pro-tein-2 (UCP1) produces and mitochondrion proton gradient uncoupling and be heat by biochemistry power conversion by making ATP, is widely regarded as the clear and definite functional label of brown fat cell.The adipose cell of expressing UCP-1 is called " heat production adipose cell " herein.In mice, the genetic defect of brown adipose tissue causes extremely obesity (Lowell etc., 1993, Nature 366:740-742), and the selectivity defect of UCP1 prevent in mice to β 3-adrenergic stimulate heat production and anti-obesity response (Inokuma etc., 2006, Am J Physiol Endocrinol Metab 290:E1014-E1021), this confirms that UCP1 is key molecules (Kozak etc. in energy expenditure and fat adjustment, 2008, Int JObes 32:S32-S38).

In the mammal from rodent to people, brown fat cell exists with the dispersion storehouse form of neonatally the most significant brown adipose tissue, and this meets the heat challenge of surviving at this age.Nearest discovery shows that these brown fat storehouses retain Thermogenesis (Nedergaard etc., 2007, Am J Physiol Endocrinol Metab 293:E444-E452 in mankind's manhood; Van Marken Lichtenbelt etc., 2009, N Engl J Med 360:1500-1508; Cypess etc., 2009, N Engl J Med 360:1509-1517), produce such tissue can be used for the treatment of benefit probability by exogenous activation.Noticeablely, a considerable amount of brown fat cell is also present in (Xue etc. in " white " fat depot during early development after birth momently, 2007, J Lipid Res 48:41-51), and (Cousin etc. can be reappeared in white adipose storehouse under given conditions in the manhood, 1992, J Cell Sci 103:931-942).Even in the mankind, limited evidence shows that brown fat cell is derivable (Lean etc., 1986, Int J Obes 10:219-227) in the manhood in white adipose storehouse.Therefore, the probability also existed is, " dispersion " heat production adipose cell can be induced to be used for the treatment of benefit in conventional white fat depot.Conventional white fatty tissue storehouse in fact showed cell rebuilds degree or phenotypical plasticity, and it does not observe (Prunet-Marcassus etc., 2006, ExpCell Res 312:727-736) in the brown fat storehouse of dispersion.

As described in an embodiment, ActRIIB-Fc fusion rotein can be used for the UCP-1 signal transduction increased in the fat depot of the mice that high fat diet is fed.Therefore, the ActRIIB derived material of ActRIIB signal transduction and other compounds are suppressed to can be used for increasing quantity and/or the activity of heat production adipose cell.The part be combined with the ActRIIB of the adjustment relating to heat production adipose cell comprises activator protein (such as activator protein A, activator protein B, activated protein c and activator protein E), muscle mass (i.e. GDF-8), GDF-3, GDF-11 and Nodal.In some respects, the present invention relates to ActRIIB polypeptide.As used herein, term " ActRIIB " refers to derive from Activin receptor IIB type (ActRIIB) albumen of any species and the family of ActRIIB associated protein.The member of ActRIIB family is all transmembrane protein usually, and the specific cytoplasmic domains of serine/threonine kinase predicted by the ligand binding ectodomain in Dai Fu cysteine district, membrane spaning domain and band forms.People ActRIIB precursor has following amino acid sequences, wherein signal peptide band underscore, and ectodomain represents with runic, is connected glycosylation site adds frame (SEQ ID NO:2) (NM_001106,512 aminoacid) with potential N-.

MTAPWVALALLWGSLWPG LLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

The above-mentioned wild-type sequence comprising native leader runs through this description and uses, as the basic sequence of the amino acid number for any different truncate to ActRIIB, mature form and variant.Term " ActRIIB polypeptide " is used in reference to the polypeptide of any naturally occurring polypeptide comprising ActRIIB family member and any variant (comprising mutant, fragment, fusant and peptide mimics form) retaining useful activity thereof.Such as, ActRIIB polypeptide comprises the polypeptide of the sequence derived from any known ActRIIB, and described known ActRIIB has the sequence having at least about 80% homogeneity with the sequence of ActRIIB polypeptide and preferably have at least 85%, 90%, 95%, 97%, 99% or higher homogeneity.

In certain embodiments, the present invention relates to solubility ActRIIB polypeptide.As described herein, term " solubility ActRIIB polypeptide " is often referred to the polypeptide of the ectodomain comprising ActRIIB albumen.Term used herein " solubility ActRIIB polypeptide " comprises any naturally occurring ectodomain of ActRIIB albumen and retains any variant (comprising mutant, fragment and peptide mimics form) of useful activity.Such as, the ectodomain of ActRIIB albumen and ligand binding be generally solubility.Be below example (SEQ ID NO:1) (116 aminoacid) of solubility ActRIIB polypeptide.

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

Other examples of solubility ActRIIB polypeptide also comprise signal sequence (see embodiment 1) except the ectodomain of ActRIIB albumen.Signal sequence can be the signal sequences native of ActRIIB or the signal sequence from another albumen, such as tissue plasminogen activator's (TPA) signal sequence or honey bee melittin (honey bee mellitin, HBM) signal sequence.

Two kinds of relevant II receptors (ActRIIA and ActRIIB) have been accredited as activator protein (Mathews and Vale, 1991, Cell 65:973-982; Attisano etc., 1992, Cell 68:97-108) and the II receptor of other BMP and GDF many.Except activator protein, ActRIIA and ActRIIB can carry out biochemical interaction (Yamashita etc. with other TGF-'beta ' family albumen (comprising BMP7, Nodal, GDF8 and GDF11) some, 1995, J.Cell Biol.130:217-226; Lee and McPherron, 2001, Proc.Natl.Acad.Sci.98:9306-9311; Yeo and Whitman, 2001, Mol.Cell 7:949-957; Oh etc., 2002, Genes Dev.16:2749-54).In specific embodiments, the present invention relates to the part (being also called ActRIIB part) with theme ActRIIB polypeptide (such as solubility ActRIIB polypeptide) antagonism ActRIIB receptor.Therefore, the compositions and methods of the invention can be used for treating the disease relevant to the abnormal activity of one or more parts of ActRIIB receptor.The exemplary part of ActRIIB receptor comprises some TGF-'beta ' family members, such as activator protein (such as activator protein A, activator protein B, activated protein c and activator protein E), GDF3, Nodal, GDF8 and GDF11.

Activator protein is dimer polypeptide growth factor and belongs to TGF-beta superfamily.There are three kinds of activator proteins (A, B and AB), it is the homodimer/heterodimer (β of two kinds of closely-related β subunits _aβ _a, β _bβ _band β _aβ _b).In TGF-beta superfamily, activator protein is unique in the multi-functional factor, it can produce at ovary and placenta cells moderate stimulation hormone, support neuronal cell survival, visual cell type actively or negatively affect cell cycle progress and at least in Amphibian embryo, induce mesodermal differentiation (DePaolo etc., 1991, Proc SocEpBiol Med.198:500-512; Dyson etc., 1997, Curr Biol.7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963).In addition, it is found that the erythroid differentiation factor (EDF) identical with activator protein A (Murata etc., 1988, PNAS, 85:2434) be separated from the people's monocytic leukemic cells stimulated.This shows that activator protein A works in bone marrow as erythropoietic natural instrumentality.In some tissues, activator protein signal transduction is by its relevant heterodimer inhibin antagonism.Such as, at follicle stimulating hormone (FSH) from hypophysis deenergized period, activator protein promotes FSH secretion and synthesis, and inhibin prevents FSH from secreting and synthesis.Scalable activator protein biological activity and/or other albumen be combined with activator protein comprise Follistatin described below (FS), Follistatin associated protein (FSRP), α ₂-macroglobulin, Cerberus and Endoglin.

BMP-7 (BMP7), also known as being osteogenic protein (oseogenic protein)-1 (OP-1), its inducing cartilage and bone formation as everyone knows.In addition, BMP7 regulates a large amount of physiological process.Especially, BMP is accredited as the crucial promotive factor (Tseng etc., 2008, Nature 454:1000-1004) of brown fat cell differentiation recently.In this study, the genetic defect of BMP7 causes almost not existing completely of the shortage of brown fat in Mus embryo and UCP1.In addition, raise BMP7 in mice by adenoviral administration and express increase brown fat quality and energy expenditure.Therefore, document shows, expection BMP7's antagonistsuch as ActRIIB polypeptide or anti-ActRIIB antibody nopromote that UCP1 expresses, brown fat cell is formed and/or brown fat cytoactive.Similar with activator protein, BMP7 and II receptor ActRIIA and ActRIIB combines.But it is heteromeric receptor complex that BMP7 and activator protein raise different I receptors.The main BMP7I receptor observed is ALK2, and activator protein exclusively combines with ALK4 (ActRIIB).BMP7 and activator protein cause different biologicallies and the different Smad approach of activation (Macias-Silva etc., 1998, J Biol Chem.273:25628-36).

GDF-3 (GDF3), be also called as Vg1-relevant 2, play an important role in fetal development and also participate in lipogenesis between the manhood.In brief, the expression of GDF3 in white adipose tissue (Weisberg etc. relevant to body weight or obesity, 2003, J ClinInvest 112:1796-1808), and the overexpression of adenovirus mediated GDF3 makes the obesity observed in the wild-type mice under high fat diet condition increase too much (Wang etc., 2004, Biochem Biophys Res Commun 321:1024-1031).Importantly, mice with GDF3 genetic defect is healthy with normal maintaining standard diet, but the basal metabolic rate (Shen etc., 2009, MolEndocrinol 23:113-123) of obesity and display increase is avoided when maintaining high fat diet.In a word, these discoveries refer explicitly to GDF3 in the obesity and the more general adjustment in obesity of diet induced.

Nodal albumen works in the body of mesoderm and endoblastic induction and formation and body early embryo generation middle shaft structure (such as heart stomach function regulating) is subsequently formed.Proved the main axle construction promoting notochord and prechordal plate of back of the body tissue in the vertebrate embryos of growing, it is raised peripheral cell and forms non-axle embryonic structure simultaneously.Nodal seems to carry out signal transduction by I type and II receptor with the born of the same parents' internal effect thing being called as Smad albumen.Nearest research supports that ActRIIA and ActRIIB is as the viewpoint (Sakuma etc., GenesCells.2002,7:401-12) of the II receptor of Nodal.It shows that Nodal part and its cofactor (such as cripto) interact with the activator protein I type of activating phosphatase Smad2 and II receptor.Nodal albumen participates in the many events important to Early vertebrates embryo, comprises mesoderm formation, front pattern development (anterior pattening) and Y-axis specialization.The evidence of experiment has proved that Nodal signal transduction activates pAR3-Lux, and a kind of previous display specific reaction is in the luciferase reporter of activator protein and TGF-β.But Nodal can not induce pTlx2-Lux, a kind of specific reaction is in the reporter molecule of bone morphogenetic protein.The direct biochemical evidence that nearest result provides is, Nodal signal transduction is mediated by activator protein-TGF-beta pathway Smad, Smad2 and Smad3.More evidence display Nodal signal transduction needs extracellular cripto albumen, makes it transduce different from activator protein signal transduction or TGF-signal beta.

GDF-8 (GDF8) is also called muscle mass.GDF8 is the negative regulator agent of skeletal muscle mass.GDF8 is high expressed in the skeletal muscle of growing and adult's skeletal muscle.In transgenic mice, GDF8 null mutation passes through obvious hypertrophy and hypertrophy sign (McPherron etc., Nature, 1997,387:83-90) of skeletal muscle.(Ashmore etc., 1974, Growth, 38:501-507 in cattle; Swatland and Kieffer, J.Anim.ScL, 1994,38:752-757; McPherron and Lee, Proc.Natl.Acad.Sci.USA, 1997,94:12457-12461; With Kambadur etc., Genome Res., 1997,7:910-915) and remarkably (Schuelke etc., N Engl J Med 2004 in people; 350:2682-8), skeletal muscle mass similar be increased in naturally occurring GDF8 suddenly change in be obvious.Study the increase (Gonzalez-Cadavid etc., PNAS, 1998,95:14938-43) that the amyotrophy relevant to HIV also showing people is attended by GDF8 protein expression.In addition, GDF8 scalable muscle specific enzyme (such as creatine kinase) generation and regulate myoblastic cell proliferation (WO 00/43781).GDF8 propetide can with ripe GDF8 domain dimer Non-covalent binding, its biologic activity of deactivation (Miyazono etc. (1988) J.Biol.Chem., 263:6407-6415; Wakefield etc. (1988) J.Biol.Chem., 263; 7646-7654; With (1990) Growth Factors, 3:35-43 such as Brown).The protein binding relevant on GDF8 or structure also suppresses other albumen of its biologic activity comprise Follistatin and may comprise Follistatin associated protein (Gamer etc. (1999) Dev.Biol., 208:222-232).

GDF-11 (GDF11), is also called BMP11, is a kind of secretory protein (McPherron etc., 1999, Nat.Genet.22:260-264).During mice develop, GDF11 expresses (Nakashima etc., 1999, Mech.Dev.80:185-189) in tail bud, appendage bud, maxillary and mandibular arch and dorsal root ganglion.GDF11 plays a part unique (Gamer etc., 1999, Dev Biol., 208:222-32) in the pattern development of mesoderm and nervous tissue.Display GDF11 is cartilage generation and myogenetic negative regulator agent (Gamer etc., 2001, Dev Biol.229:407-20) in developmental chicken limbs.The expression of GDF11 in muscle also shows it and is regulating the effect in muscle growth in the mode being similar to GDF8.In addition, the expression display GDF11 of GDF11 in brain also may have the activity relevant to neural function.Noticeablely, find that GDF11 suppresses neural and (Wu etc., 2003, Neuron.37:197-207) occurs in olfactory epithelium cell.Therefore, GDF11 can have and applies in the in vitro and in vivo of disease therapy such as muscle disease and neurodegenerative diseases (such as amyotrophic lateral sclerosis).

In some respects, the present invention relates to the purposes that the ActRIIB ligand signal in specific ActRIIB polypeptide (such as solubility ActRIIB polypeptide) any process that antagonism is relevant to ActRIIB activity is usually transduceed.Optionally, ActRIIB polypeptide of the present invention can one or more parts of antagonism ActRIIB receptor, such as activator protein (such as activator protein A, activator protein B, activated protein c and activator protein E), GDF3, Nodal, GDF8 and GDF11, and can therefore for the treatment of other diseases.

Therefore, the expection of this description uses the antagonist for treating of ActRIIB polypeptide and ActRIIB or ActRIIB part or prevents relevant disease active in heat production adipose cell or disease.ActRIIB or ActRIIB part participates in the adjustment of many critical biological processes.Such metabolic disease or the example of the patient's condition include but not limited to metabolism syndrome (being also called syndrome X), diabetes, glucose tolerance, fasting glucose lowers (impaired fasting flucose), the plasma insulin concentrations raised and insulin resistant, dyslipidemia, hyperlipemia, gluttony and polyphagia, colon cancer, carcinoma of prostate, breast carcinoma, carcinoma of endometrium and renal carcinoma, osteoarthritis, obstructive sleep apnea, cholelithiasis, cholelithiasis, hypertension, heart disease, heart rate exception and arrhythmia, myocardial infarction, congestive heart failure, coronary heart disease, coronary artery disease, angina pectoris, sudden death, PCOD, craniopharyngioma, Prader-Willi syndrome, Frohlich Cotard, GH lacks experimenter, normal variant short stature, other pathological condition of rest energy expenditure of Turner syndrome and the metabolic activity reduced with the percentages show of total fat-free quality or minimizing, such as, suffer from the child of acute lymphoblastic leukemia.Sexual and the reproductive dysfunction (such as sterile) of more examples, male hypogonadism and female hirsutism, gastrointestinal motility disease (the stomach esophageal reflux that such as obesity is relevant), respiratory disorder (such as obesity-hypoventilation syndrome or Pickwickian syndrome), cardiovascular disease, cerebral infarction, cerebral thrombosis, transient ischemic attack, inflammation (systemic inflammatory of such as vascular structure), arteriosclerosis, hypercholesterolemia, hyperuricemia, fatty liver, gout, gallbladder disease, Orthopedic Disorders and lumbago and backache (lower back pain).These diseases and disease be " exemplary treatment purposes " middle discussion below.

In the special context used in the context of the present invention and at each term, the term used in this description generally has its its ordinary meaning in the art.Description hereafter or other more local terms are discussed, so that at description the compositions and methods of the invention and how to manufacture and use in them and provide extra guidance to practitioner.According to the special context using term, the scope of any use of this term or implication will be apparent.

In view of the character measured or degree of accuracy, " about " and " being similar to " is often referred to the acceptable error degree of measured amount.Exemplary error degree is generally in 20% of the scope of set-point or value, preferably in 10%, and more preferably in 5%.

Alternatively, and especially in biology system, term " about " and " being similar to " can refer to that value is in the order of magnitude of set-point, preferably in 5 times and more preferably in 2 times.Unless otherwise indicated, herein given numerical quantities is approximate, means can imply term " about " and " being similar to " when not clearly stating.

Method of the present invention can comprise the step mutually compared between sequence, comprises comparing of wild-type sequence and one or more mutant (sequence variants).Such comparison generally includes the comparison of polymer sequences, such as, use alignment programs well known in the art and/or algorithm (such as BLAST, FLAST and MEGALIGN only lift several example).Those skilled in the art can easily recognize, in such comparison that sudden change comprises residue insertion or disappearance, sequence alignment will introduce " room (gap) " (usually being represented by short-term or " A ") in the polymer sequences not comprising insertion or deleting residues.

Term used herein " diabetes " refers to noninsulindependent diabetes (NIDDM is also called type ii diabetes).Type i diabetes or insulin-dependent diabetes (IDDM) are the results that insulin definitely lacks, and insulin is the hormone regulating glucose utilization.Type ii diabetes or insulin-dependent diabetes (i.e. noninsulindependent diabetes) occur when the insulin level in the face of normally or even raising usually, and seemingly suitably respond the result organizing anergy of insulin.Most of type ii diabetes is also fat.

In its grammatical forms all and spelling change, " homology " refers to the relation had between two albumen of " common evolutionary origin ", and albumen comprises the homologous protein of the albumen of the superfamily of the same species from organism and the different plant species from organism.Such albumen (and code nucleic acid) has sequence homology, as by their sequence similarity reflection, no matter according to percentage identities still by the existence of specific residue or die body and conservative position.

" obesity " is a kind of patient's condition that there is excessive body fat.The operational definition of obesity, based on Body Mass Index (BMI), is calculated as body weight/square metre height (kg/m ²) calculate." obesity " refers to that by diagnosis be the fat patient's condition.A kind of standard grading system is as described below is generally used for European, African, native American people or American Indian descendant, and a kind of alternative system is generally used for asian patients.According to this system, obesity is defined as to have and is more than or equal to 30kg/m ²the experimenter of other aspect health of BMI, or there is at least one have with sick experimenter and be more than or equal to 27kg/m ²the patient's condition of BMI.

In its grammatical forms all, term " sequence similarity " refers to or to share the homogeneity between the nucleotide sequence of common evolutionary origin or aminoacid sequence or the degree of consistency.

But in common use and the application, when with adverbial word, such as " highly " modifies, term " homology " can refer to sequence similarity and or may may not relate to common evolutionary origin.

2.ActRIIB polypeptide

In some respects, the present invention relates to ActRIIB variant polypeptide (such as solubility ActRIIB polypeptide).Optionally, fragment, functional variety and modified forms have the similar or identical biologic activity of its corresponding wild type ActRIIB polypeptide.Such as, ActRIIB variant of the present invention can combine with ActRIIB part (such as activator protein A, activator protein AB, activator protein B, activated protein c, activator protein E, GDF3, Nodal, GDF8 or GDF11) and suppress its function.Optionally, the growth of the tissue such as ActRIIB polypeptides for modulating such as fat, muscle, bone or cartilage.The example of ActRIIB polypeptide comprises people ActRIIB Precursor Peptide (SEQ ID NO:2) and soluble human ActRIIB polypeptide (such as SEQ ID NO:1,5,6,12,14 and 17).

The functional activity part of this description qualification ActRIIB and variant.Applicant has been found that to have by (Blood.1994 April 15 such as Hilden; 83 (8): 2163-70) the Fc fusion rotein of disclosed sequence has alanine (A64) on the position of aminoacid 64 corresponding to SEQ ID NO:2, and it has relative low affinity to activator protein with GDF-11.In contrast, in position 64 with the identical Fc fusion rotein of arginine (R64) to the affinity of activator protein and GDF-11 within the scope of the paramount picomolar concentrations of low nanomolar concentration.Therefore in this manual the sequence of band R64 is used as the wild-type reference sequence of people ActRIIB.

(Cell.1992 January 10 such as Attisano; 68 (1): 97-108) show, reduce receptor to the affinity of activator protein in the disappearance of ActRIIB ectodomain C-terminal proline knot.Relative to ActRIIB (the 20-134)-Fc comprising proline interface and complete nearly spanning domain, the ActRIIB-Fc fusion rotein " ActRIIB (20-119)-Fc " comprising the aminoacid 20-119 of SEQ ID NO:2 has the combination reduced with GDF-11 and activator protein.But even if proline interface is destroyed, ActRIIB (20-129)-Fc albumen still retains activity that is similar but that reduce a little relative to wild type.Therefore, the ActRIIB ectodomain that expection ends at amino acid/11 34,133,132,131,130 and 129 has activity, but the construct ending at 134 or 133 may have activity most.Similarly, to expect that in residue 129-134 sudden change on any one is not because large edge (margin) changes ligand binding affinity.The sudden change of P129 and P130 does not reduce ligand binding in fact, supports this viewpoint.Therefore ActRIIB-Fc fusion rotein can end at amino acid/11 09 (last cysteine) the earliest, but, expection end at 109 and 119 or form between 109 and 119 there is the ligand binding of reduction.Amino acid/11 19 conservative is poor, so be easily changed or clip.End at 128 later form reservation ligand-binding activities.End at 119 and 127 or form between 119 and 127 will have middle binding ability.Any one in these forms can desirably use, and this depends on clinical setting or experimental situation.

At the N-terminal of ActRIIB, the albumen that expection originates in before aminoacid 29 will retain ligand-binding activity.Aminoacid 29 represents first cysteine.The alanine mutation of position 24 is the glycosylation sequences that agedoite introduces N-connection, but does not affect ligand binding in fact.This proves, the sudden change in the district (corresponding to aminoacid 20-29) between signal cutting peptide and cysteine cross district is well tolerable.Especially, originate in the construct of position 20,21,22,23 and 24 by retentive activity, and expection originates in the construct also retentive activity of position 25,26,27,28 and 29.

In a word, the active part of ActRIIB comprises the aminoacid 29-109 of SEQ ID NO:2, and construct such as can originate in the residue of corresponding aminoacid 20-29 and end at the position of corresponding amino acid/11 09-134.Other examples comprise the position that originates in 20-29 or 21-29 and end at the construct of position of 119-134,119-133 or 129-134,129-133.Other examples comprise the position the construct ending at the position of 109-134 (or 109-133), 119-134 (or 119-113) or 129-134 (or 129-133) that originate in 20-24 (or 21-24, or 22-25).Also expect variant within the scope of these especially with the appropriate section of SEQ ID NO:2, to there is the variant of at least 80%, 85%, 90%, 95% or 99% homogeneity.

This description comprises the result of the analysis of synthesis ActRIIB structure, and it proves that ligand binding pocket is limited by residue Y31, N33, N35, L38-T41, E47, E50, Q53-K55, L57, H58, Y60, S62, K74, W78-N83, Y85, R87, A92 and E94-F101.Over these locations, although K74A sudden change is well tolerable, expection conservative variants will be tolerated, as the sudden change of R40A, K55A, F82A and L79 position.R40 is K in Xenopus laevis, shows will to be tolerated at this position basic amino acid.Q53 is R and is K in Xenopus laevis ActRIIB in cattle ActRIIB, and the aminoacid therefore comprising R, K, Q, N and H will be tolerated in this position.Therefore, the general formula of active A ctRIIB misfolded proteins is such albumen, it comprises aminoacid 29-109, but optionally originate in the position of 20-24 or 22-25 and end at the position of 129-134, and comprise in ligand binding pocket and be no more than 1,2,5,10 or 15 conserved amino acid and change, and position 40,53,55,74,79 and/or 82 in ligand binding pocket comprises zero, one or more non-conservation changes.Such albumen can retain more than 80% with the sequence of the aminoacid 29-109 of SEQ ID NO:2,90%, the sequence iden of 95% or 99%.Variability especially can be comprised the amino of ectodomain (as mentioned above) and carboxyl terminal and position 42-46 and 65-73 by the outer site of well tolerable binding pocket.On position 65, agedoite is changed into alanine (N65A) and in fact improve ligand binding under A64 background, and therefore expects not have adverse influence to ligand binding under R64 background.This change may get rid of the glycosylation on N65 under A64 background, therefore proves to tend to be tolerated in the remarkable change in this district.Although it is poor to change toleration to R64A, R64K is well tolerable, and therefore other alkaline residue such as H can be tolerated on position 64.

ActRIIB conservative in nearly all vertebrates is good, and wherein many sections of sequences of ectodomain are completely conservative.The many parts also high conservative be combined with ActRIIB.Therefore, provide can the understanding of reformed residue from the comparison of different vertebrate organism ActRIIB sequence.Therefore, active people ActRIIB variant can comprise from the one or more aminoacid on another kind of vertebrates ActRIIB sequence relevant position, maybe can comprise the residue similar to the residue in people or other vertebrates sequences.Following instance illustrates the method to limit active A ctRIIB variant.L46 is valine in Xenopus laevis ActRIIB, so this position can be changed, and optionally can be changed to another hydrophobic residue such as V, I or F, or non-polar residue such as A.E52 is K in Xenopus laevis, shows that this site can tolerate a large amount of changes, comprises polar residues such as E, D, K, R, H, S, T, P, G, Y and possible A.T93 is K in Xenopus laevis, shows to tolerate structure variation widely on this position, preference polar residues such as S, K, R, E, D, H, G, P, G and Y.F108 is Y in Xenopus laevis, and therefore Y or other hydrophobic group such as I, V or L should be tolerated.E111 is K in Xenopus laevis, shows that charged residues will be tolerated on this position, comprises D, R, K and H, and Q and N.R112 is K in Xenopus laevis, shows that alkaline residue is tolerated on this position, comprises R and H.The relative conservative of A of position 119 is poor, and in rodent, shows as P and show as V in Xenopus laevis, and therefore any aminoacid all should be tolerated on this position substantially.

This description proves, the glycosylation site (N-X-S/T) adding other N-connection increases the serum halflife of ActRIIB-Fc fusion rotein relative to ActRIIB (R64)-Fc form.By introducing agedoite (A24N construct) on position 24, produce the NXT sequence of giving longer half life.In the upper discovery of 42-44 (NQS) and 65-67 (NSS) other NX (T/S) sequence, but cannot by effective for the latter glycosylation by R possibly on position 64.Usually N-X-S/T sequence can be introduced in the position outside ligand binding pocket.Specially suitable site for introducing non-endogenous N-X-S/T sequence comprises aminoacid 20-29,20-24,22-25,109-134,120-134 or 129-134.Also N-X-S/T sequence can be incorporated in the joint between ActRIIB sequence and Fc or other fusion parts.By introducing N relative to the tram of existing S or T or introduce S or T on the position corresponding to existing N, introduce such site with minimum effort.Therefore, desirable the changing into of the glycosylation site that N-connects can be produced: A24N, R64N, S67N (may change with N65A and combine), E106N, R112N, G120N, E123N, P129N, A132N, R112S and R112T.Due to the protection provided by glycosylation, can will be predicted as glycosylated any S without the need to producing immunogenicity site and change into T.Similarly, be predicted as glycosylated any T and can change into S.Therefore expection changes S67T and S44T.Similarly, in A24N variant, S26T can be used to change.Therefore ActRIIB variant can comprise the glycosylation consensus sequence that one or more non-endogenous N-additionally connects.

Position L79 can be changed into activator protein-muscle mass (GDF-11) binding characteristic given and changing.L79A or L79P reduces GDF-11 and combines higher than the degree of reduction activator protein combination.L79E or L79D retains GDF-11 and combines.Significantly, L79E and L79D variant has the activator protein combination greatly reduced.Experiment in vivo shows that these inactive protein receptors retain significant ability to increase muscle quality, but display reduces the effect of its hetero-organization.These data show to obtain the desirability to the polypeptide that activator protein effect reduces and feasibility.

Described variation can be combined by different way.In addition, the result of mutagenesis procedures as herein described shows, exists and be usually of value to conservative amino acid position in ActRIIb.It is (hydrophobic that these comprise position 64 (basic amino acid), position 80 (acid or hydrophobic amino acid), position 78, and be in particular tryptophan), position 37 is (acid, and be in particular aspartic acid or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid is in particular phenylalanine or tyrosine).Therefore, in each variant disclosed herein, this description provides the amino acid framework that may guard.Other positions of guarding may be needed as described below: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidity), 98 (polarity or charged, is in particular E, D, R or K).

In specific embodiments, the fragment of the separation of ActRIIB polypeptide produces the polypeptide of the respective segments restructuring generation of the nucleic acid (such as SEQ ID NO:3 and 4) of own coding ActRIIB polypeptide and obtains by screening restructuring.In addition, the such as conventional Merrifield solid phase f-Moc of technology known in the art or t-Boc chemistry can be used to carry out chemosynthesis fragment.(recombinate or pass through chemosynthesis) and test fragment can be produced, can such as those peptidyl fragments that antagonist (inhibitor) or the agonist (activator) of ActRIIB albumen or ActRIIB part work to identify.

In specific embodiments, the functional variety of ActRIIB polypeptide have be selected from SEQID NO:1,2,5,6,12, the aminoacid sequence of 14 and 17 has the aminoacid sequence of at least 75% homogeneity.In particular instances, functional variety have be selected from SEQ ID NO:1,2,5,6,12, the aminoacid sequence of 14 and 17 has the aminoacid sequence of at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity.

In specific embodiments, the present invention's expection by modifying the structure manufacturing function variant of ActRIIB polypeptide, for such as strengthening the object such as therapeutic efficiency or stability (pot-life in such as ex vivo and body endoproteinase degradation-resistant).Also the ActRIIB polypeptide of modification can be produced, such as, by amino acid replacement, disappearance or interpolation.Such as, rational expectation can not have main impact to the biologic activity of produced molecule with isoleucine or valine displacement leucine, glutamic acid displacement aspartic acid, serine for threonine or the similar displacement (such as conservative variants) with amino acid-toamino acid relevant in structure separately.Conservative substitution is the displacement occurred in the amino acid whose family that its side chain is correlated with.Whether the change in the aminoacid sequence of ActRIIB polypeptide causes function congener, easily measures in cell by assessment modification A ctRIIB polypeptide with the aitiogenic ability of mode being similar to wild type ActRIIB polypeptide or the ability that is combined with one or more parts such as activator protein (activator protein A, activator protein B, activated protein c and activator protein E), Nodal, GDF3, GDF11 or muscle mass in the mode being similar to wild type.

In certain embodiments, the present invention expects that the specific sudden change of ActRIIB polypeptide is to change the glycosylation of polypeptide.Exemplary glycosylation site in ActRIIB polypeptide is illustrated in SEQ ID NO:2.Can select such sudden change to introduce or to eliminate one or more glycosylation site, such as O-connects or N-connects glycosylation site.The glycosylation recognition site that agedoite connects comprises by the tripeptide sequence of suitable cellular glycosylation enzymes specific recognition usually, asparagine-X-threonine (wherein " X " is any aminoacid).Also by adding to the sequence of wild type ActRIIB polypeptide or replacing one or more serine or threonine residues and make changes (for O-connection glycosylation site).Cause on the tripeptide sequence modified non-glycosylated in one of first or the 3rd amino acid position of glycosylation recognition site or the displacement of the different aminoacids on both or disappearance (and/or the aminoacid deletion on second position).Another method that ActRIIB polypeptide increases the quantity of carbohydrate portions is for by by glucosides and ActRIIB chemiluminescent polypeptide or enzymatic coupling.Depend on the conjugation pattern of use, sugar can be connected to (a) arginine and histidine; (b) free carboxy; (c) free sulfhydryl groups, the sulfydryl of such as cysteine; (d) free hydroxyl group, the such as hydroxyl of serine, threonine or hydroxyproline; (e) aromatic residue, the such as aromatic residue of phenylalanine, tyrosine or tryptophan; Or the amide groups of (f) glutamine.These methods WO87/05330 and at Aplin and Wriston (1981) CRC Crit.Rev.Biochem. disclosed in JIUYUE in 1987 11 days, state in 259-306 page, they are incorporated herein by reference.Removing be present in one or more carbohydrate portions on ActRIIB polypeptide can chemically and/or enzyme method complete.Chemical deglycosylation can comprise such as makes ActRIIB polypeptide be exposed to compound trifluoromethanesulfonic acid or equivalent compound.Although remaining complete aminoacid sequence, this process causes the cutting of great majority except connecting sugar (N-acetyl-glucosamine or N-acetylgalactosamine) or all sugar.Chemical deglycosylation is further described by (1981) Anal.Biochem.118:131 such as (1987) Arch.Biochem.Biophys.259:52 and Edge such as Hakimuddin.The enzyme action of the carbohydrate portions on ActRIIB polypeptide cuts and is completed by the various endoglycosidase described in (1987) Meth.Enzymol.138:350 such as Thotakura and exoglycosidase by using.Depend on that used expression system type can adjust the sequence of ActRIIB polypeptide as one sees fit because mammal, yeast, insecticide and plant cell all can to introduce different glycosylations graphic, these graphic aminoacid sequences by this peptide affect.Usually, will express providing in suitable glycosylated mammal cell line such as HEK293 or Chinese hamster ovary celI system for the ActRIIB albumen that uses in the mankind, but expect that other mammalian expression cell systems are also useful.

This description is also expected and is produced the method for variant, and the set of the variant especially combinatory variants of ActRIIB polypeptide, optionally comprises truncated variant; Combination mutant pond especially can be used for qualification functional variety sequence.The object of screening such combinatorial library can be the ActRIIB polypeptide variants producing the characteristic (pharmacokinetics such as changed or the ligand binding of change) such as with change.There is provided various Screening test below, and this mensuration can be used for assessing variant.Such as can screen ActRIIB polypeptide variants for the ability being combined with ActRIIB polypeptide to prevent ActRIIB part to be combined with ActRIIB polypeptide.

The activity of ActRIIB polypeptide or its variant also can be tested in based on the mensuration of cell or in vivoassay.Such as can assess ActRIIB polypeptide variants to the impact (such as UCP-1) of expression of gene participating in Adipocyte Differentiation or function.This can carry out under the existence of one or more ActRIIB ligandin (such as GDF8) that recombinate, and can turn seven cell to produce ActRIIB polypeptide and/or its variant and optionally to produce ActRIIB part.Similarly, ActRIIB polypeptide can be given mice or other animals, and one or more characteristics of adipose cell can be assessed, such as brown fat cell heat production.Similarly, any impact of activity on the growth of these cells of ActRIIB polypeptide or its variant can be tested in adipose cell, myocyte, osteocyte and neurocyte, such as, by following mensuration.This being determined at is known in the art and conventional.The impact of the reactive reporter gene monitoring of SMAD on downstream signal transduction can be used in such cell line.

The combination with the selectivity usefulness relevant to naturally occurring ActRIIB polypeptide can be produced and derive variant.When expressing from recombinant dna construct, such misfolded proteins can use in gene therapy approach.Similarly, mutation can produce the variant with half life in the born of the same parents significantly different from corresponding wild type ActRIIB polypeptide.Such as, the protein vs protein enzymatic degradation of change can be made or cause other processes of the destruction of natural A ctRIIB polypeptide or otherwise inactivation more stable or more unstable.This variant and their gene of coding can be used for by regulating the half life of ActRIIB polypeptide to change ActRIIB peptide level.Such as, short half life, can cause ofer short duration biological action, and when part can inducible expression time, can allow to be more tightly controlled intracellular restructuring ActRIIB peptide level.

In specific embodiments, ActRIIB polypeptide of the present invention, except the natural any modification be present in ActRIIB polypeptide, also can comprise post translational modification.This modification includes but not limited to acetylation, carboxylation, glycosylation, phosphorylation, esterified and acidylate.Therefore, the ActRIIB polypeptide of modification can comprise non-amino acid composition, such as Polyethylene Glycol, lipid, polysaccharide or monosaccharide and phosphate ester.This non-amino acid composition can be tested for other ActRIIB polypeptide variants as described herein on the impact of the function of ActRIIB polypeptide.When ActRIIB polypeptide is produced by the nascent form of cutting ActRIIB polypeptide in cell, post translational processing is also to the correct folding of albumen and/or function is important.Different cell (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) for activity after this translation, there is specific cell system and distinctive mechanism, and different cell can be selected to guarantee correct modification and the processing of ActRIIB polypeptide.

In some respects, the functional variety of ActRIIB polypeptide or modified forms comprise and have at least part of ActRIIB polypeptide and the fusion rotein of one or more Fusion domain.The well known examples of this Fusion domain includes but not limited to polyhistidine, Glu-Glu, glutathione-S-transferase (GST), thioredoxin, protein A, Protein G, immunoglobulin heavy chain constant region (such as Fc), maltose-binding protein (MBP) or human serum albumin.Fusion domain can be selected to give required characteristic.Such as, some Fusion domains are particularly useful for by affinity protein purification fusion rotein.In order to the object of affinity purification, use and be used for the relevant substrate of affinity chromatograph, such as glutathion-, the resin of amylase-and nickel-or cobalt-put together.Much this substrate can obtain with " test kit " form, such as Pharmacia GST purification system and with (HIS ₆) QIAexpress that uses together of fusion partner ^tMsystem (Qiagen).As another example, Fusion domain can be selected to contribute to the detection of ActRIIB polypeptide.The example in this detection architecture territory comprises various fluorescin (such as GFP) and " epitope tag ", " epitope tag " be generally specific antibody can short peptide sequence.The known epitope tag that easily can obtain monoclonal antibody specific comprises FLAG, influenza virus haemagglutinin (HA) and c-myc label.In some cases, Fusion domain has proteolytic cleavage site, such as Xa factor or thrombin, and it enables the partial digested fusion rotein of protease of being correlated with also therefore therefrom discharge recombiant protein.Can be then separated with Fusion domain by the albumen of chromatography subsequently by release.In particular preferred embodiment, ActRIIB polypeptide with make ActRIIB polypeptide in vivo stable structure territory (" stabilisation " domain) merge." stabilisation " refers to any situation increasing serum halflife, and no matter this is due to the destruction of minimizing, the renal clearance of reduction or other pharmacokinetics effects.Pharmacokinetic properties needed for the fusion of the Fc of immunoglobulin part is known much albumen being given.Similarly, required characteristic can be given with the fusion of human serum albumin.Other fusion structure field types selectable comprise multimerization (such as dimerization, four dimerizations) domain and functional domain (giving extra biological function, such as stimulated muscle growth further).

As specific example, the invention provides the fusion rotein as GDF8 antagonist, it comprises (such as GDF8 be combined) domain (such as SEQ IDNO:13) outer with the born of the same parents of Fc domain fusion.

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALH N(A)HYTQKSLSLSPGK

Optionally, Fc domain has one or more sudden change on residue such as Asp-265, lysine 322 and Asn-434.Under specific circumstances, the mutant Fc domain with these sudden changes (such as Asp-265 sudden change) one or more relative to wild type Fc domain there is reduction with the ability of Fc γ receptors bind.In other cases, the ability that the mutant Fc domain with these sudden changes one or more (such as Asn-434 sudden change) combines relative to the Fc receptor (FcRN) relevant to MHC I class that wild type Fc domain has increase.

Should be appreciated that the heterogeneity of fusion rotein can arrange by any mode consistent with required function.Such as, ActRIIB polypeptide can be arranged and make C-end facing to hetero-structures territory, or alternatively, hetero-structures territory can be arranged and make C-end facing to ActRIIB polypeptide.ActRIIB polypeptide domain and hetero-structures territory do not need adjacent in fusion rotein, and can comprise extra domain or aminoacid sequence and make C-end or N-end facing to arbitrary domain or extra domain or aminoacid sequence be included between domain.

In specific embodiments, ActRIIB polypeptide of the present invention comprises one or more modifications that can make ActRIIB stabilizing polypeptides.Such as, this proteasome degradation modified the external half life increasing ActRIIB polypeptide, the circulation half life increasing ActRIIB polypeptide or reduce ActRIIB polypeptide.This stabilisation is modified and is included but not limited to the modification (comprise and such as glycosylation site is added ActRIIB polypeptide) of fusion rotein (comprising the fusion rotein such as comprising ActRIIB polypeptide and stabilisation domain), glycosylation site and the modification (comprise and such as remove carbohydrate portions from ActRIIB polypeptide) of carbohydrate portions.With regard to fusion rotein, ActRIIB polypeptide and stabilisation domains such as IgG molecule (such as Fc domain) merge.As used herein, term " stabilisation domain " not only refers to as the Fusion domain (such as Fc) with regard to fusion rotein, and comprises non-protein modification (such as carbohydrate portions) or non-protein polymer (such as Polyethylene Glycol).

In specific embodiments, the present invention makes the unpack format of ActRIIB polypeptide and/or purified form obtain, itself and other Protein Separation, or is substantially free of other albumen.

In specific embodiments, ActRIIB polypeptide of the present invention (unmodified or modify) produces by many technology known in the art.Such as, this ActRIIB polypeptide can use standard protein chemical technology to synthesize, such as use Bodansky, M.Principles ofPeptide Synthesis, Springer Vetlag, Berlin (1993) and Grant G.A. (chief editor), Synthetic Peptides:A User ' s Guide, technology described in W.H.Freeman and Company, NewYork (1992).In addition, automated peptide synthesizer has commercially available (such as Advanced ChemTech Model 396; Milligen/Biosearch 9600).Alternatively, various expression systems (such as, escherichia coli, Chinese hamster ovary cell, COS cell, the baculovirus) restructuring of (also vide infra) known in this field can be used to produce ActRIIB polypeptide, its fragment or variant.In another embodiment, by using such as protease (such as trypsin, thermolysin, Chymotrypsin, pepsin) or paired basic amino acid converting enzyme (PACE) to digest total length ActRIIB polypeptide that is naturally occurring or restructuring generation, produce ActRIIB polypeptide that is that modify or unmodified.Computer analysis (using commercial software, such as MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used for identifying proteolytic cleavage site.Alternatively, this ActRIIB polypeptide can produce the total length ActRIIB polypeptide produced from natural existence or restructuring, such as, by standard technique known in the art, and such as chemical cleavage (such as Bromine cyanide., azanol).

3. the nucleic acid of coding ActRIIB polypeptide

In some respects, the invention provides coding any ActRIIB polypeptide (such as solubility ActRIIB polypeptide) (comprising any variant disclosed herein) separation and/or recombinant nucleic acid.Such as, following sequential coding naturally occurring people ActRIIB Precursor Peptide (SEQ ID NO:4) (nucleotide 5-1543, the 1539bp of NM_001106):

atgacggcgccctgggtggccctcgccctcctctggggatcgctgtggcccggctctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccctgctcacggtgctggcctactcactgctgcccatcgggggcctttccctcatcgtcctgctggccttttggatgtaccggcatcgcaagcccccctacggtcatgtggacatccatgaggaccctgggcctccaccaccatcccctctggtgggcctgaagccactgcagctgctggagatcaaggctcgggggcgctttggctgtgtctggaaggcccagctcatgaatgactttgtagctgtcaagatcttcccactccaggacaagcagtcgtggcagagtgaacgggagatcttcagcacacctggcatgaagcacgagaacctgctacagttcattgctgccgagaagcgaggctccaacctcgaagtagagctgtggctcatcacggccttccatgacaagggctccctcacggattacctcaaggggaacatcatcacatggaacgaactgtgtcatgtagcagagacgatgtcacgaggcctctcatacctgcatgaggatgtgccctggtgccgtggcgagggccacaagccgtctattgcccacagggactttaaaagtaagaatgtattgctgaagagcgacctcacagccgtgctggctgactttggcttggctgttcgatttgagccagggaaacctccaggggacacccacggacaggtaggcacgagacggtacatggctcctgaggtgctcgagggagccatcaacttccagagagatgccttcctgcgcattgacatgtatgccatggggttggtgctgtgggagcttgtgtctcgctgcaaggctgcagacggacccgtggatgagtacatgctgccctttgaggaagagattggccagcacccttcgttggaggagctgcaggaggtggtggtgcacaagaagatgaggcccaccattaaagatcactggttgaaacacccgggcctggcccagctttgtgtgaccatcgaggagtgctgggaccatgatgcagaggctcgcttgtccgcgggctgtgtggaggagcgggtgtccctgattcggaggtcggtcaacggcactacctcggactgtctcgtttccctggtgacctctgtcaccaatgtggacctgccccctaaagagtcaagcatctaa

Following sequential coding human soluble (born of the same parents are outer) ActRIIB polypeptide (SEQ ID NO:3) (348bp).

tctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccacc

That theme nucleic acid can be strand or double-strand.This nucleic acid can be DNA or RNA molecule.These nucleic acid can such as use in the method for the preparation of ActRIIB polypeptide, or use as direct therapeutant (such as in gene therapy method).

In some respects, the theme nucleic acid of coding ActRIIB polypeptide is also interpreted as the nucleic acid comprised for SEQ ID NO:3 variant.Variant nucleotide sequences comprises the sequence of the one or more nucleotide subsitution of difference, interpolation or disappearance, such as allelic variant; With will therefore comprise the coded sequence different from the nucleotide sequence of coded sequence shown in SEQ ID NO:4.

In specific embodiments, the invention provides to have with SEQ ID NO:3 at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity be separated or recombinant nucleic acid sequence.Those of ordinary skill in the art will recognize, with the variant of the nucleotide sequence of SEQ ID NO:3 complementation and SEQ ID NO:3 also within the scope of the invention.In further embodiment, nucleotide sequence of the present invention can be separation, restructuring and/or merge with heterologous nucleotide sequence, or in DNA library.Such as, the invention provides to have with SEQID NO:10 or 15 at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity be separated or recombinant nucleic acid sequence.

In other embodiments, nucleic acid of the present invention is also included in the nucleotide sequence of hybridizing with complementary series or its fragment of nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:3 under high stringency.As described above, those of ordinary skill in the art will readily appreciate that the appropriate stringency conditions alterable promoting DNA hybridization.Those of ordinary skill in the art will readily appreciate that the appropriate stringency conditions alterable promoting DNA hybridization.Such as, can hybridize under 6.0x sodium chloride/sodium citrate (SSC) at about 45 DEG C, then carry out the washing of 2.0xSSC at 50 DEG C.Such as, the salinity in washing step can be selected from the high stringency of the low stringency to 50 of 50 DEG C of about 2.0x SSC DEG C about 0.2x SSC.In addition, the temperature in washing step can be increased to about 65 DEG C of high stringent conditions from room temperature (about 22 DEG C) low stringency condition.Temperature and salt both alterable, or temperature or salinity can remain unchanged, and change another variable.In one embodiment, the invention provides the nucleic acid of hybridizing under the low stringency condition of room temperature 6x SSC and then washing under room temperature 2x SSC.

The nucleic acid that is separated different from nucleic acid shown in SEQ ID NO:3 due to the degeneracy of genetic code also within the scope of the invention.Such as, much aminoacid is specified by more than a triplet.Specify the codon of same amino acid or synonymous codon (such as CAU and CAC is the synonymous codon of histidine) that " silence " of the aminoacid sequence not affecting albumen can be caused to suddenly change.But, expect that the DNA sequence polymorphism really causing the aminoacid sequence of theme protein to change will be present in mammalian cell.Person of skill in the art will appreciate that, these in one or more nucleotide (can reach the nucleotide of about 3-5%) of the nucleic acid of encode specific protein make a variation because natural allelic variation can be present in the individuality of given species.Any and all this nucleotide diversities and the amino acid polymorphism that produces are within the scope of the invention.

In specific embodiments, recombinant nucleic acid of the present invention can regulate nucleotide sequence to be effectively connected with one or more in expression construct.Regulate the host cell that nucleotide sequence will be generally suitable for for expressing.Eurypalynous suitable expression vector and suitable adjustment sequence perhaps for different hosts cell are known in the art.Usually, described one or more regulate nucleotide sequence can include but not limited to promoter sequence, targeting sequencing or signal sequence, ribosome binding site, transcription initiation and terminator sequence, translation initiation and terminator sequence and enhancer sequence or activate son (activator) sequence.Constitutive promoter known in the art or inducible promoter are desired by the present invention.Promoter can be naturally occurring promoter or the combination hybrid promoter more than the element of a promoter.Expression construct can be present on intracellular episome (such as plasmid), or expression construct can be inserted in chromosome.In a preferred embodiment, expression vector comprises selected marker to allow the host cell selecting to transform.Selected marker is well known in the art, and changes with used host cell.

More of the present invention in, theme nucleic acid is comprising coding ActRIIB polypeptide and is providing in the expression vector of the nucleotide sequence regulating sequence to be effectively connected with at least one.Regulate sequence to be known in the art and be selected for the expression instructing ActRIIB polypeptide.Therefore, term regulates sequence to comprise promoter, enhancer and other expression control elements.Exemplary adjustment sequence is shown in and is set forth in Goeddel; Gene Expression Technology:Methods inEnzymology, Academic Press, San Diego, CA (1990).Such as, any one control when being effectively connected with it in multiple expression control sequenc that DNA sequence expresses can use in these carriers, to express the DNA sequence of coding ActRIIB polypeptide.This useful expression control sequenc comprises the early stage of such as SV40 and late promoter, tet promoter, adenovirus or the instant early promoter of cytomegalovirus, RSV promoter, lac system, trp system, TAC or TRC system, the T7 promoter expressed is instructed by T7 RNA polymerase, the major operator of bacteriophage lambda and promoter region, the control zone of fd coat protein, the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferments, the promoter (such as Pho5) of acid phosphatase, the promoter of yeast α-mating factor, other sequences of the polyhedron promoter of rhabdovirus system and the gene expression of known control prokaryotic cell or eukaryotic cell or its virus, and its various combination.Should be appreciated that, the design of expression vector can be depending on the factor such as the selection of such as host cell to be transformed and/or the protein types of needs expression.In addition, also should consider the copy number of carrier, control the expression of the ability of copy number and any other albumen (such as antibiotic marker) by vector encoded.

Recombinant nucleic acid of the present invention is suitable for producing in prokaryotic cell, eukaryotic cell (yeast, birds, insecticide or mammal) or the carrier of expressing in both by the gene of clone or its part being connected to.Expression vector for generation of restructuring ActRIIB polypeptide comprises plasmid and other carriers.Such as, suitable carrier comprises the plasmid with Types Below: the plasmid that the plasmid that the plasmid that the plasmid that the plasmid that pBR322 derives, pEMBL derive, pEX derive, pBTac derive and pUC are derivative, at prokaryotic cell such as expression in escherichia coli.

Some mammalian expression vectors comprise the protokaryon sequence and one or more eukaryotic transcription unit at eukaryotic expression that promote that carrier is bred in antibacterial.The carrier that pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derive is the example of the mammalian expression vector being suitable for transfecting eukaryotic cells.Some in these carriers, with the sequence modification from bacterial plasmid (such as pBR322), are selected with drug resistance to contribute to copying in prokaryotic cell and eukaryotic cell.Alternatively, the derivant of virus such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP derivative and p205) can be used for the transient expression of albumen in eukaryotic cell.The example of other viruses (comprising retrovirus) expression system can find in the description of hereafter gene therapy delivery system.The various methods used in the preparation of plasmid and in the conversion of host living beings are well known in the art.About other Suitable expression vectors and the general recombination method of prokaryotic cell and eukaryotic cell, see Molecular Cloning A Laboratory Manual, 2nd edition, chief editor Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) the 16th and 17 chapters.In some cases, by using baculovirus expression system expression recombinant polypeptide to be desirable.The carrier (such as comprising the pBlueBacIII of β-gal) that the carrier (such as pAcUW1) that example comprises the derivative carrier (such as pVL1392, pVL1393 and pVL941) of pVL, pAcUW derives of this baculovirus expression system and pBlueBac derive.

In a preferred embodiment, carrier design is used in Chinese hamster ovary celI, produce theme ActRIIB polypeptide, such as Pcmv-Script carrier (Stratagene, La Jolla, Calif), pcDNA4 carrier (Invitrogen, Carlsbad, Calif) and PCI-neo carrier (Promega, Madison, Wisc.).The cells that theme gene construct can be used for causing theme ActRIIB polypeptide to breed in culture will be obvious, such as, produce the protein being used for purification, comprise fusion rotein or misfolded proteins.

The present invention also relates to the host cell with recombination transfection, and this recombination comprises the coded sequence (such as SEQ ID NO:3,4,10 or 15) of one or more themes ActRIIB polypeptide.Host cell can be any prokaryotic cell or eukaryotic cell.Such as, ActRIIB polypeptide of the present invention can be expressed in bacterial cell (such as escherichia coli), insect cell (such as using baculovirus expression system), yeast or mammalian cell.Other suitable host cells are known to those skilled in the art.

Therefore, the invention still further relates to the method producing theme ActRIIB polypeptide.Such as, the host cell of the expression vector transfection with coding ActRIIB polypeptide can be cultivated under suitable conditions, occur to allow the expression of ActRIIB polypeptide.ActRIIB polypeptide can secretion and being separated from cell mixture and the culture medium comprising ActRIIB polypeptide.Alternatively, ActRIIB polypeptide can be retained in kytoplasm or in membrane component with in the cell lysis collected and in the albumen be separated.Cell culture comprises host cell, culture medium and other by-products.Suitable culture medium for cell culture is well known in the art.Theme ActRIIB polypeptide can use the technology for purifying protein known in the art (comprising the immunoaffinity purification of the special antibody of ion-exchange chromatography, gel permeation chromatography, ultrafiltration, electrophoresis and the use defined epitope to ActRIIB polypeptide) from cell culture medium, host cell or both be separated.In a preferred embodiment, ActRIIB polypeptide is the fusion rotein comprising the domain promoting its purification.

In another embodiment, the fusion gene of coding purification leader sequence (the N-end of the required part of the ActRIIB polypeptide of such as recombinating many-(His)/enterokinase cleavage site point sequence), can enable the fusion rotein of expression by use Ni ²⁺the affinitive layer purification of metal-resin.Then, purification leader sequence can be removed by enterokinase process subsequently, to provide the ActRIIB polypeptide (for example, see Hochuli etc., (1987) J.Chromatography 411:177 and Janknecht etc., PNAS USA 88:8972) of purification.

Technology for the preparation of fusion gene is known.In essence, the connection of various DNA fragmentations of different peptide sequence of encoding is carried out according to routine techniques, blunt end or staggered end is used to be used for connecting, restriction endonuclease digestion is to provide suitable end, optionally fill cohesive end, alkaline phosphatase treatment is connected with enzymatic to avoid unwanted connection.In another embodiment, fusion gene is synthesized by routine techniques (comprising automatization DNA synthesizer).Alternatively, the anchor primer of the complementary overhangs caused between two consecutive gene fragments can be used to carry out the pcr amplification of genetic fragment, complementary overhangs can be annealed to produce chimeric gene sequence (see such as Current Protocols in Molecular Biology subsequently, chief editor Ausubel etc., John Wiley & Sons:1992).

4. antibody and other antagonisies

Another aspect of the present invention relates to antibody and other antagonisies, comprises the albumen be combined with target disclosed herein and the nucleic acid suppressing target disclosed herein to be expressed.To use with the antagonist that the antibody of ActRIIB polypeptide competition binding can be used as ActRIIB polypeptide active with ActRIIB polypeptide (such as solubility ActRIIB polypeptide) specific reaction.Such as, by using the immunogen deriving from ActRIIB polypeptide, prepare anti-albumen/anti-peptide antiserum or monoclonal antibody (the Antibodies:ALaboratory Manual (Cold Spring Harbor Press:1988) see such as being edited by Harlow and Lane) by standard scheme.The immunogen form immunising mammals of available ActRIIB polypeptide or part, the antigen fragment that can cause antibody response or fusion rotein, such as mice, hamster or rabbit.Technology for giving albumen or peptide based immunogens comprises puts together or other technologies well known in the art with carrier.The immunogen part of ActRIIB polypeptide or part can be given under adjuvant exists.By detecting the antibody titer monitoring immunization schedule in blood plasma or serum.Using immunogen as antigen, standard ELISA or other immunoassay can be used to assess the level of antibody.

After the antigen preparation immune animal with ActRIIB polypeptide or part, can antiserum be obtained, and optionally can be separated polyclonal antibody from serum.In order to produce monoclonal antibody, can gather in the crops from the animal of immunity and producing the cell (lymphocyte) of antibody, and by standard somatic cell fusion program and immortalized cell such as myeloma cell fusion to produce hybridoma.This technology is known in the art, and comprise such as hybridoma technology (at first by Kohler and Milstein, 1975, Nature, 256:495-497 develop), human B-lymphocyte hybridoma technology (Kozbar etc., 1983, Immunology Today, 4:72) and the EBV-hybridoma technology (Cole etc. of human monoclonal antibodies, 1985, Monoclonal Antibodies andCancer Therapy, Alan R.Liss, Inc. 77-96 page).Can immunochemistry screening hybridoma for generation of the antibody reacted with ActRIIB polypeptid specificity and the monoclonal antibody of culture being separated self-contained this hybridoma.

Term used herein " antibody " is intended to comprise its fragment of also reacting with theme ActRIIB polypeptide or ligand specificity.Routine techniques can be used antibody fragmentation, and to screen the effectiveness of fragment with the identical method for complete antibody mentioned above.Such as, by producing F (ab) with pepsin antibody ₂fragment.The F (ab) of generation can be processed ₂fragment is to reduce disulphide bridges and produce Fab fragment.Antibody of the present invention be also intended to comprise there is the affinity to ActRIIB polypeptide given by least one CDR district of antibody bispecific, strand and chimeric and humanized molecule.In preferred embodiments, antibody also comprises connected labelling and can be detected (such as, labelling can be radiosiotope, fluorescent chemicals, enzyme or enzyme cofactor).

In particular preferred embodiment, antibody of the present invention is monoclonal antibody, and in specific embodiments, the present invention is used in the method producing new antibodies and can obtains.Such as, method for generation of the monoclonal antibody be combined with ActRIIB polypeptide or ligand specificity can comprise: give a certain amount of immunogenic composition comprising ActRIIB polypeptide or part of mice with the detectable immunoreation of effective stimulus, from mice, obtain the cell (such as splenocyte) that produces antibody and will cell and the hybridoma cell fusion of antibody be produced to obtain the hybridoma producing antibody, and the hybridoma testing generation antibody is to identify the hybridoma of the monoclonal antibody that generation is combined with ActRIIB polypeptide or ligand specificity.Once obtain, hybridoma can be bred in cell culture, under the cell optionally derived at hybridoma produces the condition of culture of the monoclonal antibody be combined with ActRIIB polypeptide or ligand specificity.Can from cell culture monoclonal antibody purification.

When for antibody, it is the same that adjective " with ... specific reaction " is intended to refer to usually to understand with this area, antibody has sufficient selectivity between target antigen (such as ActRIIB polypeptide) and other non target antigens, make antibody bottom line can be used for detect particular type biological sample in target antigen there is situation.In the ad hoc approach using antibody, such as treatment use, the binding specificity of higher degree can be required.Monoclonal antibody generally has the polypeptide of antigen that larger tendency (compared with polyclonal antibody) needs with effective district office and cross-linking reaction.Affect antibody: a specific feature of AI is the affinity of antibody to antigen.Although required specificity can different affinity scope realize, preferred antibody will have about 10 usually ^-6, 10 ^-7, 10 ^-8, 10 ^-9or lower affinity (dissociation constant).

In addition, for screening antibodies to identify that the technology of required antibody can affect the character of obtained antibody.Such as, if antibody is used for conjugated antigen in the solution, so test solution combines desirable.Available for test antibody and antigen interphase interaction with many different technologies of the desirable especially antibody of qualification.This technology comprises ELISA, the resonance of surperficial plasmon combines and measures (such as Biacore combination mensuration, Bia-core AB, Uppsala, Sweden), sandwich assay (such as IGEN International, Inc., the paramagnetic bead system of Gaithersburg, Maryland), Western blotting, immune precipitation determination and immunohistochemistry.

In some respects, this description provides the antibody with solubility ActRIIB polypeptide or ligand binding.Substantially can produce such antibody as described above ground, use solubility ActRIIB polypeptide or part or its fragment as antigen.The antibody of the type can be used for such as detecting the ActRIIB polypeptide in biological sample and/or the solubility ActRIIB peptide level in monitoring individuality.Under specific circumstances, the antibody be combined with solubility ActRIIB polypeptide or ligand specificity can be used for the activity regulating ActRIIB polypeptide and/or ActRIIB part, thus increases heat production adipose cell.

By using the polypeptide or its variant that comprise the bound fraction of respective propetide, suppress particular ligand such as muscle mass and GDF3.This propetide can be prepared as fusion rotein, comprises Fc fusion rotein.The example of suitable propetide is open in publication application WO 02/085306 and WO06/002387.

In addition, other associated proteins can be used, such as so-called " trapper (trap) " (such as Follistatin, FLRG, FSTL, Cerberus and Coco), soluble Type I receptor, such as ALK-7.The example of this peptide species is found in publication application WO05/115439, WO 08/109779, WO 08/067480, WO 07/109686, WO05/100563 and WO 05/025601.

Nucleic acid can be used, such as antisense or RNAi probe (it can comprise the nucleotide that naturally occurring nucleotide and non-natural exist), with the expression of any part suppressing ActRIIB or discuss herein.

5. Screening test

In some respects, the present invention relates to the purposes that theme ActRIIB polypeptide (such as solubility ActRIIB polypeptide) qualification is the agonist of ActRIIB polypeptide or the compound (material) of antagonist.Can be tested in the tissues such as such as fat, muscle, bone, cartilage and/or neuron, to assess the ability of its external regulate tissue growth by the compound of this Screening and Identification.Optionally, these compounds can test the ability assessing regulate tissue growth in its body further in animal model.

There is many methods for screening the therapeutant being carried out regulate tissue growth by targeting ActRIIB polypeptide.In specific embodiments, the high flux screening of compound can be carried out, to identify the material of the effect to fat, muscle, bone, cartilage and/or neuronic growth of interference ActRIIB mediation.In specific embodiments, carry out measuring with the suppression of screening and identification specificity or the compound reducing ActRIIB polypeptide and the such as combination of ActRIIB part (such as activator protein, GDF3, Nodal, GDF8 or GDF11) of its binding partners.Alternatively, the compound that can be used for identifying that enhancing ActRIIB polypeptide and its associated proteins (such as ActRIIB part) combine is measured.In another embodiment, by itself and the interactional ability authenticating compound of ActRIIB polypeptide.

Various mensuration form will be enough, but according to this description, the unclear in the text mensuration form described will be readily understood by one of ordinary skilled in the art.As described herein, test compounds of the present invention (material) produces by any combinational chemistry.Alternatively, motif compound can be in body or the naturally occurring biomolecule of external synthesis.Test its compound as the ability of tissue growth regulator (material) such as to pass through antibacterial, yeast, plant or other biological generation (such as natural product), chemically generation (such as micromolecule, comprises peptide mimics) or produce with recombination method.The test compounds of the present invention's expection comprises non-peptidyl organic molecule, peptide, polypeptide, peptide mimics, sugar, hormone and nucleic acid molecules.In one particular embodiment, test substances is less than about 2 for having, the organic molecule of 000 daltonian molecular weight.

Test compounds of the present invention can be used as independent discrete entities and provides, or provides in more complicated library, such as, prepared by combinatorial chemistry.These libraries can comprise such as alcohol, alkyl halide, amine, amide, ester, aldehyde, ether and other class organic compound.Test compounds being presented to test macro can using the form be separated or mixture as compound, especially in initial screening step.Optionally, compound optionally can use other compound derivings, and has the derivatization group promoting compound separation.The limiting examples of derivatization group comprise biotin, fluorescein, digoxygenin, green fluorescent protein, isotope, polyhistidine, magnetic bead, glutathione s-transferase (GST), can the cross-linking agent of photoactivation or its any combination.

In the drug screening programs in many test compounds and natural extract library, within during preset time, the quantity of the compound of research is maximized, high throughput assay is desirable.The mensuration of carrying out in cell free system (such as using purification or semipurified albumen to obtain) is preferably " mainly " screening usually, because can produce the fast Development of the change in the molecular target that they mediate by test compounds with permission and relatively easy detection.In addition, test compounds is on generally can be out in the cold in the impact of cytotoxicity or bioavailability in vitro system, measure on the contrary and mainly concentrate on the impact of medicine on molecular target, as between ActRIIB polypeptide and its associated proteins (such as ActRIIB part) binding affinity change as shown in.

Only in order to illustrate, in an exemplary Screening test of the present invention, by target compound with to be separated and the ActRIIB polypeptide of purification contacts, this polypeptide usually can with ActRIIB ligand binding, the object optionally for measuring.Then the compositions comprising ActRIIB part is added in the mixture of compound and ActRIIB polypeptide.The detection of ActRIIB/ActRIIB ligand complexes and be quantitatively provided for the method measuring effect that compound suppresses (or reinforcement) complex between ActRIIB polypeptide and its associated proteins to be formed.Effect of compound is assessed by producing dose-effect curve from the data using the test compounds of variable concentrations to obtain.In addition, blank determination can also be carried out to provide comparison base.Such as, in blank determination, will to be separated and the ActRIIB part of purification joins in the compositions comprising ActRIIB polypeptide, and the formation of quantitative ActRIIB/ActRIIB ligand complex when test compounds does not exist.Usually be appreciated that the possible mixing order of reactant can be different, and can mix simultaneously.In addition, cell extract and lysate can replace the albumen of purification to use to provide suitable cell-free assay system.

The complex detected between ActRIIB polypeptide and its associated proteins by multiple technologies is formed.Such as, detect by immunoassay or by chromatography, use the albumen of such as detectable label such as radiolabeled (such as ³²p, ³⁵s, ¹⁴c or ³h), the ActRIIB polypeptide of fluorescently-labeled (such as FITC) or enzyme labelling or the adjustment of the quantitative complex formation of its associated proteins.

In specific embodiments, the present invention expects that use fluorescence polarization determination and FRET (fluorescence resonance energy transfer) (FRET) measure the degree directly or indirectly measuring ActRIIB polypeptide and its associated proteins interphase interaction.In addition, other detection modes, such as based on detection mode (PCT prospectus WO 96/26432 and the U.S. Patent number 5 of fiber waveguide, 677,196), surperficial plasmon resonance (SPR), surface charge sensor, surface force sensor and many embodiments of the present invention are applicable to.

In addition, the present invention expects that using the trapping that interacts to measure (being also called " double cross mensuration ") qualification destroys or strengthen the material of ActRIIB polypeptide and its associated proteins interphase interaction.For example, see U.S. Patent number 5,283,317; Zervos etc., 1993, Cell 72:223-232; Madura etc., 1993, J Biol Chem 268:12046-12054; Bartel etc., 1993, Biotechniques 14:920-924; With Iwabuchi etc., 1993, Oncogene 8:1693-1696).In one particular embodiment, the present invention expects that use reverse two hybrid system identifies the compound (such as micromolecule or peptide) of dissociate ActRIIB polypeptide and its associated proteins interphase interaction.For example, see Vidal and Legrain, 1999, Nucleic Acids Res 27:919-29; Vidal and Legrain, 1999, Trends Biotechnol 17:374-81; With and U.S. Patent number 5,525,490,5,955,280 and 5,965,368.

In specific embodiments, motif compound is identified by itself and the interactional ability of ActRIIB polypeptide of the present invention.Interaction between compound and ActRIIB polypeptide can be covalency or non-covalent.Such as, this interaction can use external biochemical method (comprising photo-crosslinking, radiolabeled ligand binding and affinity chromatograph) to identify (JakobyWB etc. on protein level, 1974, Methods in Enzymology 46:1).Under specific circumstances, can in mensuration (such as detecting the mensuration of the compound be combined with ActRIIB polypeptide) the middle SCREENED COMPOUND based on mechanism.This can comprise solid phase binding events or fluid-phase binding events.Alternatively, can all report systems (such as beta galactosidase, luciferase or green fluorescent protein) by the gene transfection of coding ActRIIB polypeptide in cell, and preferably by high flux screening for library screening or the single member's screening using library.Other combinations based on mechanism can be used to measure, and the combination such as detecting free energy change measures.Can use to be fixed on hole, pearl or chip or to be caught by immobilized antibody or carry out combination by the target that capillary electrophoresis is resolved and measure.Usually colorimetric or fluorescence or the resonance of surperficial plasmon can be used to detect binding compounds.

In some respects, the invention provides the method for controlling body weight increase and obesity and material.On a molecular scale, lipocyte proliferation and differentiation are critical in the development of obesity, and it causes the generation of extra fat cell (adipose cell).Therefore, can the compound of any qualification of test in intact cell or tissue in external or body, with by measuring lipocyte proliferation or differentiation confirms that it regulates lipogenetic ability.Various method known in the art can be used for this object.Such as, ActRIIB polypeptide (such as solubility ActRIIB polypeptide) or test compounds to lipogenetic effect by measuring to the differentiation of mature fat cell measuring 3T3-L1 PECTORAL LIMB SKELETON based on (such as, dyeing the accumulation of triacylglycerol and the appearance by observing specific adipose cell labelling such as FABP (aP2/422) and PPAR γ 2 in vesicle by observing Oil Red O) in the mensuration of cell.See such as Reusch etc., 2000, MolCell Biol.20:1008-20; Deng etc., 2000, Endocrinology.141:2370-6; Bell etc., 2000, Obes Res.8:249-54.Another example based on the mensuration of cell comprises such as analyzes ActRIIB polypeptide and the effect of test compounds in adipose cell or adipocyte precursor cells (such as 3T3-L1 cell) propagation by monitoring bromodeoxyribouridine (BrdU) positive cell.See such as Pico etc., 1998, Mol Cell Biochem.189:1-7; Masuno etc., 2003, Toxicol Sci.75:314-20.

Should be appreciated that Screening test of the present invention is not only applicable to the variant of theme ActRIIB polypeptide and ActRIIB polypeptide, and be applicable to comprise the agonist of ActRIIB polypeptide or ActRIIB signal transduction and any test compounds of antagonist.In addition, these Screening test can be used for drug targets confirmation and quality control object.

6 exemplary treatment purposes

In specific embodiments, the disease that compositions of the present invention (such as ActRIIB polypeptide) can be used for treating or prevention is relevant to the abnormal activity of ActRIIB polypeptide and/or ActRIIB part (such as activator protein or GDF8) or the patient's condition.In specific embodiments, the invention provides by giving the ActRIIB polypeptide mentioned above individuality in need for the treatment of effective dose is treated or prevents individual method.These methods are especially for animal and the more specifically therapeutic of people and prophylactic treatment.

As used herein, the therapeutic agent of " prevention " disease or disease refers to such compound, it is in statistics sample, relative to untreated control sample, reduce the incidence rate of disease or the patient's condition in the sample for the treatment of, or relative to untreated control sample, delay the outbreak of one or more symptoms of disease or the patient's condition or reduce its order of severity.Term used herein " treatment " comprises the prevention of specifying the patient's condition or improvement or elimination that the patient's condition has occurred.

As indicated in this paper, ActRIIB-Fc promotes the expression of the albumen UCP1 of uncoupling in mediation mitochondrion, causes metabolism enlivens or heat production fatty tissue.Therefore, compositions disclosed herein can be used for treating various diseases, such as brown adipose tissue or brown fat cell lack, metabolism syndrome (being also called syndrome X), diabetes, hyperlipemia, hypercholesterolemia, disease of eating too much at one meal and polyphagia, hypertension, arteriosclerosis (coronary artery disease or coronary heart disease), myocardial infarction, congestive heart failure, cerebral infarction, cerebral thrombosis, respiratory disorder (such as Pickwickian syndrome), colon cancer, carcinoma of prostate, breast carcinoma, carcinoma of endometrium and renal carcinoma, growth hormone deficiency experimenter, normal variant short stature, Turner syndrome, with other pathological condition of the rest energy expenditure of the metabolic activity reduced with the percentages show of total fat-free quality or minimizing, such as suffers from the child of acute lymphoblastic leukemia.

In specific embodiments, compositions of the present invention (such as solubility ActRIIB polypeptide) is for promoting formation and/or the activity of heat production adipose cell.As described above, the brown fat cell in the discrete brown adipose tissue of heat production and white adipose tissue comprises the mitochondrion of great expression Uncoupling pro-tein-2 (UCP).There is high heat to take in and the energy intake of surplus can not be converted into heat by the individuality lacking brown fat cell, and be therefore forced to store untapped biochemistry energy, usually used as the white adipose tissue of hypertrophy.In body, the function of blocking-up or one or more ActRIIB parts (such as GDF8) of antagonism effectively can increase the Thermogenic Activity of brown fat cell in the discrete brown fat cell bank be distributed in white adipose tissue.The method is confirmed by data shown in this article and supports, wherein to show in the protein induced white adipose of ActRIIB-Fc UCP1 and expresses, increases overall health and form and improve the metabolism state in the mice of high fat diet.

In specific embodiments, compositions of the present invention (such as solubility ActRIIB polypeptide) is as the part treat of metabolism syndrome (being also called syndrome X and insulin resistance syndrome), and metabolism syndrome is the disease of risk and the combination of risk factor that increase development cardiovascular disease and type ii diabetes.Most patients is insulin resistant that is old, fat, sitting and that have to a certain degree.Middle part (abdominal part or internal organs) obesity is this syndromic marked feature.

In relevant embodiment, solubility ActRIIB polypeptide of the present invention and other compositionss can be used as the part treat of type ii diabetes (being also called noninsulindependent diabetes or maturity-onset diabetes), and the feature of type ii diabetes is the hyperglycemia when insulin resistant and relative insulin deficiency.In diabetes, complicated and polyfactorial metabolism changes the damage and function damage that usually cause many organs (most important is cardiovascular system).Type ii diabetes is usually relevant to obesity (abdominal part or visceral obesity), hypertension, hypercholesterolemia and metabolism syndrome.The important risk factor of type ii diabetes comprises the life style of ageing, high fat diet and sitting.

In the embodiment that other are relevant, solubility ActRIIB polypeptide of the present invention and other compositionss can be used as atherosclerotic part treat, atherosclerosis is the chronic inflammatory patient's condition, and wherein arterial wall is thickening due to the accumulation of fat depot (being commonly called speckle).Atherosclerotic risk factor comprise the life style of ageing, diabetes, dyslipoproteinemia, obesity (abdominal part or visceral obesity) and sitting.

Solubility ActRIIB polypeptide also can be used for lipodystrophy disease, and it is often relevant to metabolism syndrome.Severe insulin opposing can result from mode of inheritance and obtain the lipodystrophy of form, the lipodystrophy that human immunodeficiency virus (HIV) is relevant in the patient that latter instance comprises with antiretroviral therapy treatment.

Theme ActRIIB polypeptide also can be used as slowing down or the therapeutant developed that prevents obesity.The method is confirmed by data shown in this article and supports, wherein shows ActRIIB-Fc albumen and improves metabolism state in the mice of high fat diet.

In other embodiments, the invention provides for regulating body fat content in animal and being used for the treatment of or preventing the compositions of the patient's condition and the method for the patient's condition related to this and health risk especially related to this.According to the present invention, regulate (controls) body weight can refer to reduce or put on weight, reduce or put on weight speed that the speed that increases or increase or reduction lose weight, also can comprise and maintain or significantly do not change body weight (such as affecting for the outside or inside that not so can increase or reduce body weight) energetically.One embodiment of the invention relate to and regulate body weight by giving animal in need (such as people) ActRIIB polypeptide.

In a specific embodiment, the present invention relates to for reducing body weight in animal and/or reducing the method and compound that in animal, body weight increases, more specifically, method and the compound of obesity in the patient being used for the treatment of or improving and be in obesity risk or suffer from obesity is related to.In another embodiment, the present invention relates to the method being used for the treatment of the animal (such as suffering from the animal of exhaustion syndrome) that can not increase or retain body weight and compound.Such method is effective to putting on weight, or effective with loss to reduction body weight, or effective to improving low to unacceptable (such as unhealthy) body weight disease that is relevant or that caused by it.

As indicated in WO 2006/012627 and WO 2008/097541, compound stimulated muscle growth disclosed herein.Therefore, these compounds can be particularly useful in the disease of overlapping muscle and metabolic function exception or disease.

In specific embodiments, compositions of the present invention (such as solubility ActRIIB polypeptide) is as the part treat of muscular dystrophy.Term " muscular dystrophy " refer to by skeletal muscle and sometimes cardiac muscle and respiratory muscle weaken gradually and fail sign one group of degenerative muscle disease.Muscular dystrophy is the genetic diseases characterized by the progressive muscular atrophy from the minor variations in muscle and myasthenia.Along with the degeneration that muscle is passed in time, the muscle strength of people declines.In addition, the muscle quality of decline and the physical exertion of minimizing promote to take in the imbalance between energy intake and energy expenditure, cause excess energy to save as white adipose tissue unhealthyly.The available exemplary muscular dystrophy comprising the Regimen Chemotherapy of theme ActRIIB polypeptide comprises: Duchenne's dystrophy (DMD), becker's dystrophy (BMD), Ai-De muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (being also called blue for muscular dystrophy), myotonic dystrophy (MMD) (being also called SteinertShi disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy (CMD).

Duchenne's dystrophy (DMD) is described at 1860s first by French neuropathist Guillaume BenjaminAmand Duchenne.Becker's dystrophy (BMD) is named with the German doctor Peter Emil Becker describing this variant of DMD in nineteen fifties first.DMD is the one in modal holandric inheritance disease, and it affects 1/3, the boy of 500.When being positioned at the dystrophin gene on the chromosomal galianconism of x and damaging, DMD occurs.Because male only carries an X chromosome copy, so they only have a dystrophin gene copy.When lacking dystrophin, muscle is easily impaired in the cycle period of contraction and diastole.Although compensate by regeneration at the early stage muscle of disease, Muscle progenitor cells is unable to catch up with ongoing damage later, and healthy muscle is replaced by non-functional fibrofatty tissue.

BMD is suddenlyd change by the difference in dystrophin gene and causes.BMD patient has some dystrophins, but itself otherwise quantitatively inadequate, or of poor quality.Have muscle that the protection of some dystrophins suffers from the patient of BMD from picture trouble DMD crowd muscle serious or degenerate rapidly.

Such as, nearest research shows that the function of blocking-up or elimination GDF8 (a kind of ActRIIB part) in body effectively can treat at least some symptom in DMD and BMD patient.Therefore, theme ActRIIB polypeptide can be used as GDF8 inhibitor (antagonist) and works, and blocks the alternative approach of the function of DMD and BMD patient GDF8 and/or ActRIIB in constituting body.The method is confirmed by data shown in this article and supports, wherein shows the muscle quality in ActRIIB-Fc albumen increase muscular dystrophy mouse model.

Similarly, theme ActRIIB polypeptide provides the effective ways increasing and need muscle quality in the other diseases of muscle growth.Such as ALS is also called Lu Gelike (Lou Gehrig) family name's disease (motor neuron), and be a kind of chronic, the CNS disease that can not cure and cannot stop, it is attacked motor neuron, attacks the CNS composition connecting brain and skeletal muscle.In ALS, motor neuron worsens and final death, although and the brain of people has been still Full Featured and sensitive usually, and the order of motion never arrives muscle.The most people suffering from ALS is between 40-70 year.First motor neuron weakened is the motor neuron of leading to arm or lower limb.The people suffering from ALS has walk problem, and they may fall thing, fall down, speak ambiguous and laugh at uncontrollably or cry.Muscle in most hind leg due to need not and start atrophy.This muscle weakness will become and make people weak, and people is by needs wheelchair or become and can not leave bed activity.Most of ALS patient dies from respiratory failure or complication (as pneumonia) assisted by respirator, 3-5 from disease occurs.The method is confirmed by data shown in this article and supports, wherein shows ActRIIB-Fc albumen and improves the outward appearance of the mouse model of ALS, muscle quality and life-span.

The muscle quality of the increase of ActRIIB polypeptid induction also can to the benefits subjects suffering from muscle-wasting diseases.Gonzalez-Cadavid etc. (the same) report, GDF8 express in people with fat-free quality inversely related, and losing weight of the male of the expression that increases of GDF8 gene and trouble AIDS exhaustion syndrome is relevant.By suppressing the function of GDF8 in AIDS patient, even without eliminating completely, also can alleviate at least some symptom of AIDS, therefore significantly improving the quality of life of AIDS patient.

Muscle decay syndrome, along with the muscle loss of ageing is also usually relevant with metabolism syndrome, diabetes, arteriosclerosis, the metabolism patient's condition that dyslipidemia is relevant with other ages.The muscle quality of ActRIIB polypeptid induction also can be decayed syndromic benefits subjects to suffering from muscle.

7 pharmaceutical compositions

In specific embodiments, compound of the present invention (such as ActRIIB polypeptide) and pharmaceutically acceptable carrier are prepared.Such as, ActRIIB polypeptide can give separately or composition as pharmaceutical preparation (therapeutic combination) gives.Motif compound preparation can be used for giving for people or veterinary any convenient manner.

In specific embodiments, Therapeutic Method of the present invention comprises local, whole body or gives compositions partly as implantation or device.When administered, yes for the therapeutic combination used in the present invention not containing pyrogen, physiologically acceptable form.In addition, compositions desirably can be encapsulated or inject for delivery to target tissue site (such as bone, cartilage, muscle, fat or neuron) with viscous form, such as, there is the site of tissue injury.Topical can be suitable for wound healing and tissue repair.Material useful in the treatment of non-ActRIIB polypeptide is also optionally included in compositions mentioned above, in the method for the invention can alternatively or additionally with motif compound (such as ActRIIB polypeptide) simultaneously or sequentially to give.

In specific embodiments, compositions of the present invention can comprise and one or more therapeutic compound (such as ActRIIB polypeptide) can be delivered to target tissue site, provide structure optionally can by the resorbent substrate of health for tissue in growing.Such as, substrate can provide the slow release of ActRIIB polypeptide.Such substrate can be formed for other materials implanting medical applications by current.

The selection of host material is based on biocompatible, biological degradability, mechanical performance, appearance (cosmetic appearance) and interfacial property.The concrete purposes of theme composition will limit the preparation be applicable to.The potential substrate of compositions can be biodegradable and chemically limits calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and condensing model.Other potential materials are biodegradable and biologically clearly defined, such as bone or dermal collagen.Other substrate are made up of pure protein or extracellular matrix components.Other potential substrate are biological nondegradable and chemically limit, such as, sinter hydroxyapatite, bio-vitric, aluminate or other potteries.Substrate above-mentionedly can mention that the combination of the material of type forms by any, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.Bioceramic can be changed in the composition, such as, change and process to change aperture, granularity, grain shape and biological degradability in phosphorus calcium aluminate (calcium-aluminate-phosphate).

In specific embodiments, method of the present invention can orally give, such as with capsule, cachet, pill, tablet, lozenge (uses the substrate of seasoning, be generally sucrose and arabic gum or tragakanta), powder, the form of granule, or be the solution in waterborne liquid or on-aqueous liquid or suspensoid, or be oil-in-water or water-in-oil liquid Emulsion, or be elixir or syrup, or be that pastille (uses inert base, such as gelatin and glycerol, or sucrose and arabic gum) and/or be collutory etc., respectively comprise the material of scheduled volume as active component.Bolus, electuary or paste can also give medicament.

For in the solid dosage forms (capsule, tablet, pill, dragee, powder, granule etc.) of oral administration, one or more therapeutic compound of the present invention can be mixed with one or more pharmaceutically acceptable carriers, this carrier is such as sodium citrate or calcium hydrogen phosphate and/or any following carrier: (1) filler or supplement (extender), such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binding agent, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or arabic gum; (3) wetting agent, such as glycerol; (4) disintegrating agent, such as agar, calcium carbonate, Rhizoma Solani tuber osi or tapioca, alginic acid, some silicate and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption enhancer, such as quaternary ammonium compound; (7) such as wetting agent, such as hexadecanol and glyceryl monostearate; (8) absorbent, such as kaolin and bentonite; (9) lubricant, such as Pulvis Talci, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate and composition thereof; (10) coloring agent.With regard to capsule, tablet and pill, pharmaceutical composition also can comprise buffer agent.The solid composite of similar type also can be used as filler in soft capsule and hard filling capsule, and the excipients used is as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol etc.

Liquid dosage form for oral administration comprises pharmaceutically acceptable Emulsion, microemulsion, solution, suspensoid, syrup and elixir.Except active component, liquid dosage form also can comprise inert diluent usually used in this field, such as water or other solvents, solubilizing agent and emulsifying agent, fatty acid ester that such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, oil (being especially Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Semen Maydis oil, germ oil, olive oil, Oleum Ricini and Oleum sesami), glycerol, oxolane alcohol, Polyethylene Glycol and Pyrusussuriensis are smooth and composition thereof.Except atent solvent, Orally administered composition also can comprise adjuvant, such as wetting agent, emulsifying agent and suspending agent, sweeting agent, correctives, coloring agent, aromatic and antiseptic.

Except active ingredient beyond the region of objective existence, suspensoid also can comprise suspending agent, such as ethoxylation i-octadecanol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline Cellulose, partially aluminium hydroxide (aluminum metahydroxide), bentonite, agar and tragakanta and composition thereof.

Some compositions disclosed in this description can locally give skin or mucosa.Topical formulations also can comprise known to one or more in skin or the effective many kinds of substance of stratum corneum penetration promoter.These example is 2-Pyrrolidone, METHYLPYRROLIDONE, dimethyl acetylamide, dimethyl formamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxine and azone.Also can comprise other material to make formulation aesthetics can accept.These example is fat, wax, oil, dyestuff, aromatic, antiseptic, stabilizing agent and surfactant.Also can comprise keratolytic agent such as known in the art those.Example is salicylic acid and sulfur.

Dosage form for local or transdermal administration comprises powder, spray, ointment, paste, cream, lotion, gel, solution, patch and inhalant.Can aseptically by reactive compound and pharmaceutically acceptable carrier and with any antiseptic, buffer agent or the propellants that may need.Except motif compound of the present invention (such as ActRIIB polypeptide), ointment, paste, cream and gel also can comprise excipient, such as animal and plant fat, oil, wax, paraffin, starch, tragakanta, cellulose derivative, Polyethylene Glycol, silicone, bentonite, silicic acid, Pulvis Talci and zinc oxide or its mixture.

Except motif compound, powder and spray also can comprise excipient, such as the mixture of lactose, Pulvis Talci, silicic acid, aluminium hydroxide, calcium silicates and Silon or these materials.Spray can comprise conventional propellant in addition, such as Chlorofluorocarbons (CFCs) and volatile unsubstituted hydrocarbon, such as butane and propane.

In specific embodiments, be applicable to the combination that the pharmaceutical composition of parenteral can comprise one or more ActRIIB polypeptide and one or more pharmaceutically acceptable sterile isotonic aqueouss or non-aqueous solution, dispersion, suspensoid or Emulsion or can redissolve before use for the sterile powder of aseptic injectable solution or dispersion, it can comprise antioxidant, buffer, antibacterial, the solute making the blood of preparation and intended recipient isotonic or suspending agent or thickening agent.The suitable aqueous that can use in pharmaceutical composition of the present invention and the example of nonaqueous carrier comprise water, ethanol, polyhydric alcohol (such as glycerol, propylene glycol, Polyethylene Glycol etc.) and suitable mixture, vegetable oil (such as olive oil) and injection organic ester (such as ethyl oleate).Suitable mobility can be maintained, such as by use capsulating material (such as lecithin), in dispersion situation by maintain needed for granularity, and by use surfactant.

Compositions of the present invention also can comprise adjuvant, such as antiseptic, wetting agent, emulsifying agent and dispersant.By comprising various antibacterial agent and antifungal (such as Nipagin ester, methaform, phenol sorbic acid etc.), prophylaxis of microbial effect can be ensured.By isotonic agent, such as sugar, sodium chloride etc. comprise also may be desirable in the composition.In addition, by comprising the material of delayed absorption, such as aluminum monostearate and gelatin, cause the prolongation of injectable drug form to absorb.

Should be appreciated that by the doctor in charge, dosage will consider that the many factors of the effect changing present subject matter compound (such as ActRIIB polypeptide) determines.Many factors will depend on disease to be treated.

In specific embodiments, the present invention is also provided for the gene therapy producing ActRIIB polypeptide disclosed herein or other compounds in body.Such treatment realizes its therapeutic effect by the introducing of ActRIIB polynucleotide sequence being had in the cell or tissue as disease cited hereinabove.Recombinant expression carrier (such as embedded virus) or dispersion system of colloid can be used, complete sending of ActRIIB polynucleotide sequence.Treatment for ActRIIB polynucleotide sequence is sent and is preferably used target liposomes.

The various viral vector that can be used for gene therapy teaching herein comprise adenovirus, herpesvirus, vaccinia virus or preferred RNA viruses (such as retrovirus).Preferably, retroviral vector is Mus or the retroviral derivant of fowl.The example that can insert the retroviral vector of single exogenous gene includes but not limited to Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), MuMTV (MuMTV) and rous sarcoma virus (RSV).Many other retroviral vectors can mix multiple gene.All these carriers are transferable or mix the gene of selected marker, make the cell can identifying and produce transduction.By connecting such as sugar, glycolipid or protein, retroviral vector is made to have target specificity.Preferred targeting realizes by using antibody.Person of skill in the art will appreciate that, specific polynucleotides sequence can be inserted in reverse transcription virus gene group or with viral tunicle and be connected, and sends to allow the target-specific of the retroviral vector comprising ActRIIB polynucleotide.In a preferred embodiment, carrier targeting bone, cartilage, muscle or neuronal cell/tissue.

Alternatively, by the calcium phosphate transfection of routine, use the plasmid direct transfection tissue culture cells of encoding retrovirus structural gene gag, pol and env.Then with these cells of vector plasmid transfection comprising target gene.Retroviral vector is discharged in culture medium by gained cell.

Another targeted delivery systems of ActRIIB polynucleotide is dispersion system of colloid.Dispersion system of colloid comprises macromolecule complex, nanocapsule, microsphere, pearl and the system (comprising oil in water emulsion, micelle, the micelle of mixing and liposome) based on lipid.The preferred colloid system of the present invention is liposome.Liposome is artificial membrane vesicle, and it can be used as in vitro and in vivo delivery vector.RNA, DNA and complete virion can be encapsulated in aqueous interior and be delivered to cell (see such as Fraley etc., Trends Biochem.Sci., 6:77,1981) with biologic activity form.The method using liposome vectors to be used for effective gene transfer is known in the art, see such as Mannino etc., and Biotechniques, 6:682,1988.The combination of the compositions of liposome normally phospholipid, usually and steroid (particularly cholesterol) combine.Also other phospholipid or other lipids can be used.There is situation in what the physical property of liposome depended on pH, ionic strength and bivalent cation.

The example of lipid useful in the production of liposome comprises phosphatidyl compounds, routine phosphatidyl glycerol, phosphatidylcholine, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, sphingolipid, cerebroside and ganglioside.The phospholipid of illustration comprises PC, Dioctonoyl pnosphotidyl choline and DSPC.The targeting of liposome can based on such as organ specificity, cell-specific and organelle-specificity, and be also known in the art.

Illustration

Now generally describe the present invention, more easily will understand the present invention with reference to following examples, included embodiment only for the object of the illustration of particular of the present invention and embodiment, is not intended to limit the present invention.

The generation of embodiment 1.ActRIIB-Fc fusion rotein

Applicant builds solubility ActRIIB fusion rotein, and this fusion rotein has and the joint (three glycine aminoacid) of the ectodomain of people ActRIIB of people or mice Fc domain fusion and minimum between them.Construct is called ActRIIB (20-134)-hFc and ActRIIB (20-134)-mFc.

Purification is from the ActRIIB-hFc of Chinese hamster ovary celI system (SEQ ID NO:5) as follows

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT GGGTHTCPPCPAPFLLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ActRIIB (20-134)-hFc and ActRIIB (20-134)-mFc albumen is expressed in Chinese hamster ovary celI system.Consider three kinds of different targeting sequencings:

(i) honey bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ IDNO:7)

(ii) tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:8)

(iii) natural: MGAAAKLAFAVFLISCSSGA (SEQ ID NO:9)

Institute's preferred form of this uses TPA targeting sequencing and has following unprocessed aminoacid sequence:

MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT GGGTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK(SEO ID NO：17)

This polypeptide encodes (SEQ ID NO:10) by following nucleic acid sequence:

The N-end sequencing of the material that Chinese hamster ovary celI produces discloses main sequence-GRGEAE (SEQ ID NO:11).Notably, other constructs reported in document are initial with-SGR... sequence.

Complete purification by a series of column chromatography steps, such as, comprise the following column chromatography of any order of three kinds or more: Protein A Chromatography, Q agarose gel chromatography, phenyl Sepharose chromatography, size exclusion chromatography and cation-exchange chromatography.Available virus filtration and buffer agent have exchanged purification.

Also can at HEK293 cell and COS cells ActRIIB-Fc fusion rotein.Although it is active to provide muscle in body to build from the material of all cells system and rational condition of culture to albumen, observe physical variation, may be relevant with cell line selection and/or condition of culture.

The generation of embodiment 2:ActRIIB-Fc mutant

Applicant creates a series of sudden change in the ectodomain of ActRIIB, and these mutant proteins are produced as the soluble fusion protein between extracellular ActRIIB and Fc domain.Background ActRIIB-Fc fusion rotein has following sequence (Fc part has underscore) (SEQ ID NO:12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT GGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Difference sudden change (comprising N-terminal and C-terminal truncate) is introduced in background ActRIIB-Fc albumen.Based on the data shown in embodiment 1, if expection TPA targeting sequencing is expressed, these constructs are by missing N-terminal serine.In ActRIIB ectodomain, sudden change is produced by PCR mutation.After PCR, by Qiagen column purification fragment, by SfoI and AgeI digestion also gel-purified.These fragments are connected to (see WO2006/012627) in expression vector pAID4, make to produce the fusion chimera with human IgG1 upon connection.Be transformed into after in bacillus coli DH 5 alpha, choosing colony DNA isolation.For Mus construct (mFc), replace human IgG1 with Mus IgG2a.Examine the sequence of all mutants.

In HEK293T cell, all mutants are produced by transient transfection.Generally speaking, in 500ml rotator, by HEK293T cell with 6 × 10 ⁵cell/ml sets up and overnight incubation in Freestyle (Invitrogen) culture medium of 250ml volume.Second day, with DNA: PEI (1: 1) these cells of complex process of the whole DNA concentration of 0.5ug/ml.After 4 hours, 250ml culture medium is added and by cell culture 7 days.By screwing off cell and concentrated results conditioned medium.

Use multiple technologies (comprising such as protein A post) purified mutant body, and with low pH (3.0) glycine buffer eluting.After the neutralization, for PBS dialysis mutant.

Also in Chinese hamster ovary celI, mutant is produced by similar method.

Measure in combination and/or test mutant in bioassay.In some instances, measure with the albumen of conditioned medium instead of purification.Variant is described in such as disclosed patent application WO 06/012627 and WO 08/097541.Such variant can use in method as herein described.

Impact on white adipose tissue heat production character in the mice that embodiment 3:ActRIIB (20-134)-hFc feeds in high fat diet

To applicants studied in the male mice of feeding in high fat diet ActRIIB-Fc to the impact of brown fat cell and other metabolic end product (metabolic endpoint).10 week age, C57BL/6 mice was body weight coupling, and with ActRIIB (20-134)-hFc (n=10) or Tris buffer saline (TBS) carrier (n=7) with 10mg/kg twice subcutaneous therapy weekly, continue 60 days.During this period, mice unrestrictedly enjoys the standard chow that the diet comprising 58% fat replaces comprising 4.5% fat.At the end of research, collect epididymal adipose tissues pad, and use quantitative RT-PCR (reverse transcriptase polymerase chain reaction) to measure the level of the mRNA of coding Uncoupling pro-tein-2 (UCP1), UCP1 is the labelling of Thermogenesis in the brown fat cell of document sufficient proof, be distributed in (Cousin etc. in white adipose storehouse brown fat cell dispersal, 1992, JCell Sci 103:931-942).

ActRIIB (20-134)-hFc treatment causes the noticeable metabolic effect of a group.In the mice of high fat diet, ActRIIB (20-134)-hFc makes UCP1mRNA level in epididymal adipose tissues increase close to 9 times of (Fig. 1 compared to carrier; P < 0.05), consider that C57BL/6 mice shows the induction seriously weakened of UCP1 in crucial white adipose storehouse and brown fat cell compared to other mice germlines, this increases to the impressive especially effect (Guerra etc. of one, 1998, J Clin Invest 102:412-420; Xue etc., 2007, J Lipid Res48:41-51).ActRIIB (20-134)-hFc also produces 30% of useful serum free fatty acid concentration and reduces (P < 0.001).Importantly, the beneficial effect that the rise of UCP1 forms health with ActRIIB (20-134)-hFc, as measured in baseline and the 48th day by nuclear magnetic resonance, NMR (NMR).Under high fat diet condition, total fat mass increases to 3 times in the contrast of vehicle treatment during these 48 days, and this increase is decreased 40% by ActRIIB (20-134)-hFc treatment.To the 48th day, it is the body weight of 26% in the mice that total fat mass is treated at ActRIIB (20-134)-hFc, be 39% by contrast in control mice, and be the body weight of 64% in the mice treated at ActRIIB-Fc of fat-free mass's quality, be 55% by contrast in control mice.Therefore, under the condition of high fat diet, net result is more healthy health composition.

Embodiment 4: the impact on the heat production character of white adipose tissue in the mice that modification A ctRIIB (the 25-131)-hFc of truncate feeds in high fat diet

In research mentioned above (embodiment 3), modification A ctRIIB (the 25-131)-hFc that applicant also have studied truncate under high fat diet condition on the impact of the heat production character of white adipose tissue and other metabolic end product.

Applicant uses fusion rotein ActRIIB (the 25-131)-hFc (Figure 13-14) as created truncate above about the same preamble sequence as described in ActRIIB (20-134)-hFc and method.After expressing in Chinese hamster ovary celI, the maturation protein of purification shows sequence (SEQ ID NO:6) under having:

With ActRIIB (25-131)-hFc or Tris buffer saline (TBS) carrier with 10mg/kg twice subcutaneous therapy C57BL/6 mice in 10 week age weekly, continue 60 days.During this period, mice unrestrictedly enjoys the standard chow that the diet comprising 58% fat replaces comprising 4.5% fat.Another group mice maintaining standard chow diet also uses TBS vehicle treatment and as diet control.

Under high fat diet condition, the Histological change in the white adipose tissue that ActRIIB (25-131)-hFc treatment initiation is consistent with Thermogenesis and gene expression pattern.As shown in FIG. 2, the histological examination of epididymis white fat shows, ActRIIB (25-131)-hFc reduces fat and drips size and the formation causing the multilocular adipose cell bunch of the mark being brown fat.In addition, the immunohistochemical analysis of this tissue discloses, and because ActRIIB (25-131)-hFc treats, extensively there is kytoplasm induction (Fig. 2) of UCP1 in multilocular adipose cell and unilocular adipose cell.

With these Histological changes are remarkable changes in the expression of crucial heat production and Metabolism regulation gene in epididymis white fat, as measured by quantitative RT-PCR.In the mice of high fat diet, ActRIIB (25-131)-hFc makes UCP1 mRNA level in-site increase above 60 times (Fig. 3) compared to carrier, this is the impressive especially change of one, because as described above, this mice germline shows the induction seriously weakened of UCP1 in crucial white adipose storehouse and brown fat cell compared to other mice germlines.In addition, ActRIIB (25-131)-hFc treats increases coding sirtuin SIRT-1 (silent message regulator 2, homologue 1) the level (Fig. 4) of mRNA, SIRT-1 defends the energy-sensitive of the metabolic impairment of being induced by high fat diet to dominate regulator (deacetylase) (Pfluger etc., 2008, Proc Natl Acad SciUSA 105:9793-9798) and be indicated as fatty acid transfer important control (Rodgers etc., 2008, FEBS Lett 582:46-53).Significantly, ActRIIB (25-131)-hFc treats the level (Fig. 5) of the mRNA also increasing coding PGC-1 α (Peroxisome proliferator activator receptor-γ coactivator-1 α), PGC-1 α is the target of the SIRT-1 of document sufficient proof, it then controls brown adipose tissue Mitochondria biology and occurs and the necessary polygenic expression (Uldry etc. perhaps of Thermogenesis, 2006, Cell Metab, 3:333-341).Notably, show the heat production program that the forced expression induction of PGC-1 α in white adipocyte comprises the gene expression of UCP1, very similar in brown fat cell (Hansen etc., 2006, Biochem J 398:153-168).In this research, ActRIIB (25-131)-hFc makes the PGC-1 α gene expression in the white adipose tissue under high fat diet condition return to and the indistinguishable level of the mice of fed standard chow (Fig. 5).

Other change relevant to treatment forms the expression pattern that changes in white adipose tissue and associates with significant between useful hormone with metabolic effects.Therefore, in epididymis white fat, ActRIIB (25-131)-hFc increases coding Foxo-1 and (comprises a point jaw frame, albumen O subfamily-1) the level (Fig. 6) of mRNA, Foxo-1 is transcription factor, the target of SIRT-1 and the crucial derivant (Qiao etc., 2006, J Biol Chem281:39915-39924) of adiponectin expression.Adiponectin, a kind of hormone of adipose-derived, its concentration change is contrary with fat mass/obesity, plays important insulin-sensitizing effect (Yamauchi etc., 2001, Nat Med 7:941-946 in target tissue; Maeda etc., 2002, Nat Med8:731-737; Kadowaki etc., 2005, Endocr Rev 26:439-451).Induce consistent with Foxo-1mRNA, ActRIIB (25-131)-hFc treats the level (Fig. 7) increasing adiponectin mRNA in epididymis white fat and the circulation composition (Fig. 8) increasing adiponectin.Importantly; these change the sane minimizing with circulation insulin (Fig. 9), triglyceride, free fatty, high density lipoprotein (HDL) and low density lipoprotein, LDL (LDL) in the mice treated at ActRIIB (25-131)-hFc, cause the normalization of these parameters nearly all.Finally, above-mentioned impact, with the beneficially altering in health composition, measures in baseline and the 48th day as passed through nuclear magnetic resonance, NMR (NMR).Particularly, in the contrast of the vehicle treatment under high fat diet condition, total fat mass increases to 3 times during these 48 days, and ActRIIB (25-131)-hFc treatment makes this increase decrease similar 40%.In a word, ActRIIB (25-131)-hFc treatment under high fat diet condition causes: the Histological change in 1) consistent with Thermogenesis white adipose tissue and gene expression pattern, 2) beneficially altering on a large scale in hormone and metabolizing parameters, and 3) the health composition that improves.

Impact on brown fat storehouse in the mice that embodiment 5:ActRIIB (25-131)-mFc feeds in high fat diet

In another research, applicants studied modification A ctRIIB (the 25-131)-mFc of truncate under high fat diet condition to the impact of brown fat storehouse character in omoplate.9 week age, C57BL/6 mice was with ActRIIB (25-131)-mFc (n=20) or Tris buffer saline (TBS) carrier (n=10) with 10mg/kg twice subcutaneous therapy weekly, continued 60 days.Give the beginning 7 days before starting, mice unrestrictedly enjoys the standard chow that the diet comprising 58% fat replaces comprising 4.5% fat.Another group mice (n=10) maintaining standard chow diet also uses TBS vehicle treatment and as diet control.

Compared with standard diet, high fat diet produces some significant changes between the omoplate of brown adipose tissue in storehouse, and ActRIIB (25-131)-mFc treatment reverses each of these changes completely or to a great extent.Particularly, high fat diet causes the remarkable expansion in storehouse between omoplate and from red to pink bright its color (Figure 10).The reduction (Figure 12) doubling the density in (Figure 11) and brown fat storehouse of the expansion reflection quality of this diet induced.The storehouse density of one subgroup mice (n=4/ group) is measured by micro--computed tomography (microCT), total body fat percentage of described mice, as measured by nuclear magnetic resonance, NMR (NMR), closest to cell mean, (all mices are scanned by NMR.Under any circumstance, ActRIIB (25-131)-mFc treatment reverses the brown fat quality (Figure 11) of diet induced and the change of density (Figure 12) completely, reverses the size in the storehouse of diet induced and the change (Figure 10) of color to a great extent simultaneously.These results show, under high fat diet condition, ActRIIB (25-131)-mFc recovers character that may be relevant to healthy brown fat function to a great extent or completely, and the total size therefore reducing brown fat storehouse along with it improves the quality of brown fat.

In a word, these data show that solubility ActRIIB-Fc fusion rotein can be used as the antagonist by the ligand signal transduction of TGF-family, to increase formation and/or the activity of heat production brown fat cell, and therefore treat and take in because of high heat the metabolism patient's condition that increases the weight of and potentially also treat other patient's condition.

Be incorporated to by reference

The all public publications mentioned herein and patent are incorporated to by the mode quoted in full, as showing by each indivedual public publication or patent specifically and be individually incorporated to by reference.

Although discussed the particular of theme, above-mentioned description has been exemplary instead of restrictive.According to the review of this description and following claim, many changes will become apparent those skilled in the art.Four corner of the present invention should determine with reference to claim (four corner together with its equivalent) and description (change together with such).

Claims (32)

1. for increasing a method for heat production adipose cell in patient in need, described method comprise give effective dose be selected from following compound:

A. the polypeptide having the aminoacid sequence of at least 90% homogeneity with the sequence of the aminoacid 29-109 of SEQ ID NO:2 is comprised; With

B. by under stringent hybridization condition with the polypeptide of the nucleic acid coding of the nucleic acid hybridization of SEQ ID NO:3.

2. the process of claim 1 wherein that described polypeptide is dimer.

3. the method for claim 1 or 2, wherein said polypeptide comprises the fusion rotein with the part of ActRIIB allos.

4. the method for claim 3, the constant domain of wherein said polypeptide and immunoglobulin merges.

5. the method for claim 4, the Fc partial fusion of wherein said polypeptide and immunoglobulin.

6. the method for claim 5, wherein said immunoglobulin is human IgG1.

7. the method any one of claim 1-6, wherein said polypeptide comprises the sequence of SEQ IDNO:5 or 6.

8. the method any one of claim 1-7, wherein said patient has metabolic disease.

9. the method any one of claim 1-7, wherein said patient has muscle disease and metabolic disease.

10. the method any one of claim 1-9, wherein said polypeptide comprises the aminoacid sequence having at least 95% homogeneity with the sequence of the aminoacid 29-109 of SEQID NO:2.

The method of 11. claim 10, wherein said polypeptide comprises the aminoacid sequence having at least 97% homogeneity with the sequence of the aminoacid 29-109 of SEQ ID NO:2.

The method of 12. claim 11, wherein said polypeptide comprises the aminoacid sequence having at least 99% homogeneity with the sequence of the aminoacid 29-109 of SEQ ID NO:2.

Method any one of 13. claim 1-9, wherein said polypeptide comprises the aminoacid sequence having at least 90% homogeneity with the sequence of the aminoacid 25-131 of SEQID NO:2.

The method of 14. claim 13, wherein said polypeptide comprises the aminoacid sequence having at least 95% homogeneity with the sequence of the aminoacid 25-131 of SEQ ID NO:2.

The method of 15. claim 14, wherein said polypeptide comprises the aminoacid sequence having at least 97% homogeneity with the sequence of the aminoacid 25-131 of SEQ ID NO:2.

The method of 16. claim 15, wherein said polypeptide comprises the aminoacid sequence having at least 99% homogeneity with the sequence of the aminoacid 25-131 of SEQ ID NO:2.

Method any one of 17. claim 1-16, the UCP-1 expression giving to promote in the adipose cell of the patient treated of wherein said compound.

The method of 18. claim 17, wherein increases described UCP-1 and expresses in white adipose tissue.

19. 1 kinds of methods for increasing heat production adipose cell in patient in need, described method comprise give effective dose be selected from following compound:

A. the antagonist of ActRIIB;

B. the antagonist of muscle mass;

C. the antagonist of activator protein;

D. the antagonist of GDF11;

E. the antagonist of Nodal; With

F. the antagonist of GDF3.

The method of 20. claim 19, wherein said compound is the antagonist of ActRIIB.

The method of 21. claim 20, the antagonist of wherein said ActRIIB is selected from: the antibody be combined with ActRIIB and with coding ActRIIB nucleic acid hybridization and suppress the nucleic acid that ActRIIB produces.

The method of 22. claim 19, wherein said compound is the antagonist of muscle mass.

The method of 23. claim 22, the antagonist of wherein said muscle mass is selected from: the antibody be combined with muscle mass, with the coding nucleic acid hybridization of muscle mass and the nucleic acid suppressing muscle mass to produce and the polypeptide comprising myostatin pro peptide or its variant.

The method of 24. claim 19, wherein said compound is the antagonist of activator protein.

The method of 25. claim 24, wherein said compound is the antagonist of the activator protein being selected from activator protein A, activator protein B, activated protein c and activator protein E.

The method of 26. claim 24 or 25, the antagonist of wherein said activator protein is selected from: the antibody be combined with activator protein and suppress the nucleic acid that activator protein produces with the nucleic acid hybridization of ciphering activation albumen.

The method of 27. claim 19, wherein said compound is the antagonist of GDF3.

The method of 28. claim 27, the antagonist of wherein said GDF3 is selected from: the antibody be combined with GDF3, with the coding nucleic acid hybridization of GDF3 and the nucleic acid suppressing GDF3 to produce and the polypeptide comprising GDF3 propetide or its variant.

The method of 29. claim 19, wherein said compound is the antagonist of GDF11.

The method of 30. claim 29, the antagonist of wherein said GDF11 is selected from: the antibody be combined with GDF11, with the coding nucleic acid hybridization of GDF11 and the nucleic acid suppressing GDF11 to produce and the polypeptide comprising GDF11 propetide or its variant.

The method of 31. claim 30, wherein said compound is the antagonist of Nodal.

The method of 32. claim 31, the antagonist of wherein said Nodal is selected from: the antibody be combined with Nodal, with the coding nucleic acid hybridization of Nodal and the nucleic acid suppressing Nodal to produce and the polypeptide comprising Nodal propetide or its variant.